JP5173428B2 - Ovr110 antibody compositions and methods of use - Google Patents

Description

  This patent application claims the benefit of priority from US Provisional Patent Application No. 60 / 626,817, filed Nov. 10, 2004, the teachings of which are incorporated herein by reference in their entirety.

The present invention relates to anti-Ovr110 antibody compositions and methods for killing ovarian cancer cells, pancreatic cancer cells, lung cancer cells or breast cancer cells that express Ovr110.

Background of the Invention
Ovarian Cancer Ovarian cancer is the fourth most common cause of female cancer deaths in the United States, with more than 23,000 new cases and approximately 14,000 deaths predicted for 2001. Shridhar, V. et al., Cancer Res. 61 (15): 5895-904 (2001); Memarzadeh, S. & Berek, JS, J. Reprod. Med. 46 (7): 621-29 (2001). The American Cancer Society (ACS) predicts in 2004 about 25,580 new cases of ovarian cancer and that ovarian cancer will cause about 16,090 deaths in the United States . ACS website: cancer with the extension .org of the world wide web. The number of female deaths per year is greater for ovarian cancer than for all other gynecological malignancies combined. The incidence of ovarian cancer in the United States is estimated at 14.2 per 100,000 women per year, with ovarian cancer dying of 9 women per 100,000 per year. In 2004, about 70-75% of new diagnoses will be stage III and IV carcinomas with an estimated 5-year survival rate of about 15%. Jemal et al., Annual Report to the Nation on the Status of Cancer, 1975-2001, with a Special Feature Regarding Survival. Cancer 2004; 101: 3-27. The incidence of ovarian cancer is a global concern, and new cases are estimated to be estimated at 191,000 cases each year. Runnebaum, IB & Stickeler, E., J. Cancer Res. Clin. Oncol. 127 (2): 73-79 (2001). Unfortunately, women with ovarian cancer are usually asymptomatic until the disease has metastasized. Because no effective screening for ovarian cancer is available, about 70% of diagnosed women have advanced stage cancer with a 5-year survival rate of about 25-30%. Memarzadeh, S. & Berek, JS, supra; Nunns, D. et al., Obstet. Gynecol. Surv. 55 (12): 746-51. Conversely, women diagnosed with early stage ovarian cancer enjoy a significantly higher survival rate. Werness, BA & Eltabbakh, GH, Int'l. J. Gynecol. Pathol. 20 (1): 48-63 (2001). Although our understanding of the etiology of ovarian cancer is incomplete, the results of extensive studies in this area point to a combination of age, genetic characteristics, reproductive and dietary / environmental factors. ing. Age is an important risk factor in the development of ovarian cancer: while the risk of developing ovarian cancer before age 30 is low, the incidence of ovarian cancer increases linearly between 30 and 50 years of age, It then rises at a slower rate, with the highest incidence being women in their 70s. Jeanne M. Schilder et al, Heriditary Ovarian Cancer:. Clinical Syndromes and Management, in Ovarian Cancer 182 (.. Stephen C. Rubin & Gregory P. Sutton eds, 2 d ed 2001).

With regard to genetic factors, the family history of ovarian cancer is the most important risk factor in the development of the disease, which depends on the number of affected families, the relatives of the family to the woman, especially in the first degree. Relatives are affected by the disease. Same as above. Mutations in several genes are associated with ovarian cancer, including BRCA1 and BRCA2, both of which have important effects in the development of breast cancer, and both are associated with hereditary nonpolyposis colon cancer. HMSH2 and hMLH1 are included. Katherine Y. Look, Epidemiology, Etiology, and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCA1 located on chromosome 17 and BRCA2 located on chromosome 13 are tumor suppressor genes involved in DNA repair; mutations in these genes are associated with about 10% of ovarian cancers. 171-72; Schilder et al., 185-186 above. hMSH2 and hMLH1 are associated with DNA mismatch repair and are located on chromosomes 2 and 3, respectively; approximately 3% of hereditary ovarian carcinomas are reported to be due to mutations in these genes. Look, supra 173; Schilder et al., Supra 184, 188-89.

Reproductive factors are also associated with an increased or decreased risk of ovarian cancer. Late menopause, maternity and early menarche are all associated with an increased risk of ovarian cancer. Schilder et al., 182 above. One theory hypothesis is that these factors increase the number of ovulatory cycles in a woman's life, leading to “continuous ovulation”, which is a major cause of mutations to the ovarian epithelium. Conceivable. Ibid .; Laura J. Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic Ovarian Cancer, in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). Mutations can be explained by the fact that ovulation results in destruction and repair of the epithelium and requires increased cell division, which increases the likelihood of undetected mutations. Same as above. Support for this theory can be found in the fact that pregnancy, lactation and the use of oral contraceptives all suppress ovulation, but they confer a protective effect on the development of ovarian cancer. Same as above.

  Among dietary / environmental factors, an association between high intakes of animal fat or red meat and ovarian cancer appears to be related, while preventing free radical formation and maintaining normal cell differentiation Antioxidant vitamin A, which helps to do so, can provide a protective effect. Look, 169 above. Reports are also related to asbestos and hydrous magnesium trisilicate (talc), which can be present in septa and sanitary napkins. 169-70, same as above.

While current screening procedures for ovarian cancer show certain utility, they are extremely limited in their diagnostic capabilities and are particularly urgent in the early stages of cancer progression, where the disease is typically still asymptomatic The problem is most easily addressed. Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998); Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Werness & Eltabbakh, supra. Commonly used screening tests include birectal vaginal examination, radioimmunoassay to detect CA-125 serum tumor markers, and transvaginal ultrasonography. Burdette, 166 above. Currently, CA-125 is the only clinically approved serum marker used in ovarian cancer. CA-125 has been found to increase in many serous cancers, but only in half of women with early disease. The major clinical application of CA125 is the monitoring of successful treatment or detection of recurrence in women who have been treated for ovarian cancer. Markman M. The Oncologist; 2: 6-9 (1997). The use of CA125 as a screening marker is limited because it is often increased even in women with benign diseases such as endometriosis. Thus, whether used alone or in combination with CA125, there is a significant need for new serum markers that are more sensitive and specific for the detection of ovarian cancer. Bast RC. Et al., Early Detection of Ovarian Cancer: Promise and Reality in Ovarian Cancer . Cancer Research and Treatment Vol 107 (Stack MS, Fishman, DA, eds., 2001).

  Internal examination does not provide an adequate number of initial diagnoses, and other methods are not accurate enough. Same as above. One study reported that only 15% of patients with ovarian cancer were diagnosed as having the disease at the time of the patient's internal examination. Look, 174 above. Furthermore, the CA-125 test tended to show false positives in pre-menopausal women and was reported to have low predictive value in post-menopausal women. 174-75. Transvaginal ultrasonography is currently the preferred procedure for screening ovarian cancer, but it cannot reliably identify benign and malignant tumors, and ovarian size is normal In some cases, primary peritoneal malignancy or ovarian cancer cannot be found. Schilder et al., 194-95 above. Although genetic diagnostics for mutations in the BRCA1, BRCA2, hMSH2 and hMLH1 genes are currently available, these tests may be too expensive for some patients and are false negative or Indeterminate results can occur. Schilder et al., 191-94 above.

  Furthermore, current efforts are focused on identifying panels of biomarkers that can be used in combination. Bast RC Jr., J Clin Oncol 2003; 21: 200-205. Other markers currently evaluated as potential ovarian serum markers that could be one of a multi-marker panel to improve the detection of ovarian cancer are HE4; mesothelin; Kallikrein 5, 8, 10 and 11; and prostasin. Urban et al. Ovarian cancer screening Hematol Oncol Clin North Am. 2003 Aug; 17 (4): 989-1005; Hellstrom et al. "The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma", Cancer Res. 2003 Jul 1; 63 (13): 3695-700; Ordonez, " Application of mesothelin immunostaining in tumor diagnosis "[Application of mesothelin immunostaining in tumor diagnosis], Am J Surg Pathol. 2003 Nov; 27 (11): 1418-28; Diamandis EP et al., Cancer Research 2002; 62: 295- 300; Yousef GM et al., Cancer Research 2003; 63: 3958-3965; Kishi T et al., Cancer Research 2003; 63: 2771-2774; Luo LY et al., Cancer Research 2003; 63: 807-811; Mok SC et al., J Natl Cancer Inst 2001; 93 (19): 1437-1439.

Ovarian cancer staging by surgical exploration is important in determining the course of treatment and management of the disease. AJCC Cancer Staging Handbook 187 (Irvin D. Fleming et al. Eds., 5 ^th ed. 1998); Burdette, supra 170; Memarzadeh & Berek, supra; Shridhar et al., Supra. Stage classification is done with reference to a classification system developed by the International Federation of Gynecology and Obstetrics. David H. Moore, Primary Surgical Management of Early Epithelial Ovarian Carcinoma, in Ovarian Cancer 203 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al. Eds., 188 above. Stage I ovarian cancer is limited to the ovary and is characterized by tumor growth consisting of three sub-stages. Same as above. In substage IA, tumor growth is limited to one ovary, there is no tumor on the outer surface of the ovary, the ovarian capsule is intact, and malignant cells are not present in ascites or peritoneal lavage fluid. Same as above. Substage IB is identical to A1, except that tumor growth is limited to both ovaries. Same as above. Substage IC indicates the presence of tumor growth limited to one or both ovaries and includes one or more of the following features: capsule rupture, tumor growth on the surface of one or both ovaries , And malignant cells present in ascites or peritoneal lavage fluid. Same as above.

  Stage II ovarian cancer refers to tumor growth and pelvic invasion involving one or both ovaries. Same as above. Substage IIA includes invasion and / or dissemination into the uterus and / or fallopian tube and no malignant cells in ascites or peritoneal lavage fluid, while substage IIB invades other pelvic organs and tissues And again no malignant cells in the ascites or peritoneal lavage fluid. Same as above. Substage IIC includes pelvic infiltration similar to IIA or IIB, but includes malignant cells in ascites or peritoneal lavage fluid. Same as above.

  Stage III ovarian cancer involves tumor growth in one or both ovaries and has peritoneal metastasis across the pelvis and / or metastasis in regional lymph nodes as confirmed by microscopy. Same as above. Substage IIIA is characterized by microscopic peritoneal metastasis outside the pelvis, and substage IIIB includes peritoneal metastasis visible to the naked eye outside the pelvis, with a maximum dimension of 2 cm or less. Same as above. Substage IIIC is identical to IIIB except that the metastases are greater than 2 cm in maximum dimension and may include metastases to regional lymph nodes. Same as above. Finally, stage IV shows the presence of distant metastases except for peritoneal metastases. Same as above.

  While surgical staging is the standard for evaluating current management and treatment of ovarian cancer, it has significant drawbacks, including invasiveness of procedures, potential complications, and inaccuracy. The possibility of being included. Moore, 206-208, 213 above. In view of these limitations, alternative stages can be obtained by understanding the different gene expression in various stages of ovarian cancer and by obtaining various biomarkers to help better assess the progression of the disease. Attention has been directed to developing a classification method. Vartiainen, J. et al., Int'l J. Cancer, 95 (5): 313-16 (2001); Shridhar et al. Supra; Baekelandt, M. et al., J. Clin. Oncol. 18 (22 ): 3775-81.

Treatment of ovarian cancer typically involves a multifaceted attack, and surgical intervention serves as the basis for the treatment. Dennis S. Chi & William J. Hoskins, Primary Surgical Management of Advanced Epithelial Ovarian Cancer, in Ovarian Cancer 241 (Stephen C. Rubin & Gregory P. Sutton eds, 2d ed. 2001). For example, in the case of epithelial ovarian cancer, which accounts for approximately 90% of cases of ovarian cancer, the treatment typically consists of: (1) Total abdominal hysterectomy, bilateral salpingo-oophorectomy, Omentech Tumor reduction surgery including omentectomy and lymphadenectomy, followed by (2) adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin. Eltabbakh, GH & Awtrey, CS, Expert Op. Pharmacother. 2 (10): 109-24. Despite an 80% clinical response rate to adjuvant therapy, most patients experience tumor recurrence within 3 years of treatment. Same as above. Some patients may also receive a second tumor reduction surgery and / or secondary chemotherapy. Memarzadeh & Berek, above.

From the above it is clear that the procedures used to detect, diagnose, monitor, stage, predict and prevent recurrence of ovarian cancer are critical to the prognosis of the patient. Furthermore, while current procedures are useful in each of these analyses, they are limited due to their specificity, sensitivity, invasiveness and / or their cost. Thus, highly specific and sensitive procedures that work by detecting novel markers in cells, tissues or body fluids with minimal invasiveness and at a reasonable cost are highly desirable.
Therefore, to determine whether a person is likely to develop ovarian cancer, to determine whether ovarian cancer has metastasized, to diagnose ovarian cancer, to monitor disease progression, and to classify ovarian cancer Therefore, there is a great need for more sensitive and accurate methods for imaging ovarian cancer. There is also a need for better treatment of ovarian cancer.

Breast cancer, also called breast cancer, is the second most common cancer among women and accounts for one-third of cancers diagnosed in the United States. One in nine women develops breast cancer in her lifetime, about 192,000 new cases of breast cancer are diagnosed annually, and about 42,000 deaths occur. Bevers, Primary Prevention of Breast Cancer, in Breast Cancer , 20-54 (Kelly K Hunt et al., Ed., 2001); Kochanek et al., 49 Nat'l. Vital Statistics Reports 1, 14 (2001). Breast cancer is extremely rare in young women under the age of 20, and very rare in women under the age of 30. The incidence of breast cancer increases with age and becomes noticeable by the age of 50 years. Non-Latin American white women have the highest incidence of breast cancer, and Korean women have the lowest incidence. Increased prevalence of genetic mutations BRCA1 and BRCA2 that promote breast cancer and other cancers is found in Ashkenazi Jews. African American women have the highest mortality for breast cancer in these same groups (31 per 100,000), while Chinese women account for 11 per 100,000 Has the lowest mortality rate. Men can also have breast cancer, which is extremely rare. In the United States, 217,440 new cases of breast cancer and 40,580 deaths from breast cancer are estimated in 2004. (American Cancer Society website: cancer with the extension .org of the world wide web). Except for cases with associated genetic factors, the exact cause of breast cancer is unknown.

In the treatment of breast cancer, detection and risk assessment are of considerable importance because accurate and early staging of breast cancer has a significant impact on survival. For example, breast cancer detected at an early stage (stage T0, described below) has a 5-year survival rate of 92%. Conversely, if the cancer is not detected until the late stage (ie stage T4 (IV)), the 5-year survival rate drops to 13%. AJCC Cancer Staging Handbook pp. 164-65 (Irvin D. Fleming et al., 5 ^th ed. 1998). Some detection techniques, such as mammography and biopsy, are accompanied only by discomfort, expense and / or increased radiation and are only prescribed for patients at high risk for breast cancer.

  Current methods for predicting or detecting breast cancer risk are not optimal. One method for predicting the relative risk of breast cancer is by testing patient risk factors and exploring aggressive diagnosis and treatment plans for high-risk patients. The risk of breast cancer in a patient is increased age, premature birth, family history of breast cancer, personal history of breast cancer, early menarche, late menopause, late age of first full term pregnancy, preproliferative breast disease, early age Positively associated with breast irradiation and personal history of malignancy. Lifestyle factors such as fat consumption, alcohol consumption, education and socio-economic status have also been associated with increased incidence of breast cancer, but no direct causation has been established. While these risk factors are statistically significant, their weak association with breast cancer has limited their usefulness. Most women who develop breast cancer do not have any of the risk factors described above in addition to the risks that come with aging. NIH Publication No. 00-1556 (2000).

  Current screening methods for detecting cancer, such as breast self-examination, ultrasound and mammography, have drawbacks that reduce their effectiveness or prevent their widespread adoption. While breast self-examination is useful, in the early stages where the tumor is small and difficult to detect by palpation, breast cancer detection is unreliable. Ultrasonic measurements require skilled operators at increased costs. While mammography is highly sensitive, it is prone to overdiagnosis in detecting lesions suspected of being malignant. There is also a fear for the radiation used in mammography, since previous irradiation to the chest is a factor associated with increased breast cancer incidence.

  At this point, there is no appropriate method for breast cancer prevention. Current methods of breast cancer prevention include prophylactic mastectomy (mastectomy performed prior to cancer diagnosis) and chemoprevention (chemotherapy prior to cancer diagnosis) for women at high risk for breast cancer Even in the midst of these treatments, these treatments are limited. Bevers, above.

Many genetic markers have been associated with breast cancer. Examples of these markers include carcinoembryonic antigen (CEA) (Mughal et al., JAMA 249: 1881 (1983)), MUC-1 (Frische and Liu, J. Clin. Ligand 22: 320 (2000)). HER-2 / neu (Haris et al., Proc. Am. Soc. Clin. Oncology 15: A96 (1996)), uPA, PAI-1, LPA, LPC, RAK and BRCA (Esteva and Fritsche, Serum and Tissue Markers for Breast Cancer, in Breast Cancer , 286-308 (2001)). These markers have problems with limited sensitivity, low correlation and false negatives, thus limiting their use in initial diagnosis. For example, BRCA1 gene mutations are useful as an indicator of increased risk for breast cancer, while only 6.2% of breast cancers are BRCA1 positive, limiting their use in cancer diagnosis. Malone et al., JAMA 279: 922 (1998). See also Mewman et al., JAMA 279: 915 (1998) (only 3.3% correlation).

There are four main classifications of breast cancer, depending on the site of appearance and the degree of disease development.
I. In situ ductal carcinoma (DCIS): Malignant transformation of ductal epithelial cells remaining in its normal location. DCIS is a purely local disease and does not metastasize.
II. Invasive ductal carcinoma (IDC): A malignant tumor of ductal epithelial cells that penetrates the basement membrane into the supporting tissue of the breast. The IDC may eventually extend to other parts of the body.
III. In situ lobular carcinoma (LCIS): A malignant tumor that occurs in a single lobule of the breast and does not invade through the lobule wall, which generally remains local.
IV. Infiltrating lobular carcinoma (ILC): A malignant tumor that begins in a single lobule of the breast and penetrates the lobule wall directly into the adjacent tissue. This invasion across the leaflet wall can infiltrate lymphatic vessels and blood vessels and spread to distal sites.

  To determine prognosis and treatment, these four breast cancer types were staged by major tumor size (T), lymph node involvement (N), and presence of metastases (M). By definition, DCIS represents a local stage I disease, but other forms of breast cancer can range from stage II to stage IV. In addition, there are additional prognostic factors that guide surgical and medical intervention. The most common are the total number of involved lymph nodes, ER (estrogen receptor) status, Her2 / neu receptor status and histological class.

Breast cancer is diagnosed in the appropriate stage category, recognizing that different treatments are more effective for different stages of cancer. Stage TX indicates that the primary tumor cannot be evaluated (ie, the tumor has been removed or the breast tissue has been removed). Stage T0 is characterized by abnormalities such as hyperplasia, but is not accompanied by a trace of the primary tumor. Stage Tis is characterized by Paget disease of the nipple without in situ tumor, intraductal carcinoma, lobular carcinoma in situ or tumor. Stage T1 (I) is characterized by having a tumor of 2 cm or less in maximum dimension. Within stage T1, Tmic indicates a microinvasive less than 0.1 cm, T1a indicates a 0.1-0.5 cm tumor, T1b indicates a 0.5-1 cm tumor, and T1c is Tumors from 1 cm to 2 cm are shown. Stage T2 (II) is characterized by a tumor that is 2 cm to 5 cm in maximum dimension. Tumors larger than 5 cm are classified as stage T3 (III). Stage T4 (IV) represents a tumor of any size with invasion to the chest wall or skin. Within stage T4, T4a indicates tumor invasion into the chest wall, T4b indicates skin edema or ulceration of the skin satellite nodules confined to the skin of the breast or the same breast, and T4c is between T4a and T4b. Combinations are shown and T4d indicates inflammatory carcinoma. AJCC Cancer Staging Handbook pp. 159-70 (Irvin D. Fleming et al., Eds., 5 ^th ed. 1998). In addition to standard stage classification, breast tumors can be classified by their estrogen receptor and progesterone receptor protein status. Fisher et al., Breast Cancer Research and Treatment 7: 147 (1986). Additional pathological conditions such as the HER2 / neu condition may also be useful. Thor et al., J. Nat'l. Cancer Inst. 90: 1346 (1998); Paik et al., J. Nat'l. Cancer Inst. 90: 1361 (1998); Hutchins et al., Proc. Am Soc. Clin. Oncology 17: A2 (1998) .; and Simpson et al., J. Clin. Oncology 18: 2059 (2000).

  In addition to staging the primary tumor, breast cancer metastasis to regional lymph nodes can also be staged. Stage NX indicates that the lymph nodes cannot be evaluated (ie removed previously). Stage N0 indicates no regional lymph node metastasis. Stage N1 shows metastasis to a movable ipsilateral axillary lymph node. Stage N2 shows metastases to ipsilateral axillary lymph nodes fixed to each other or to other structures. Stage N3 shows metastasis to ipsilateral internal mammary lymph nodes. Same as above.

  Stage determination has a potential prognostic value and provides a basis for designing an optimal therapy. Simpson et al., J. Clin. Oncology 18: 2059 (2000). In general, pathological staging of breast cancer is preferred over clinical staging because it provides a more accurate prognosis. However, clinical stage classification is more preferred if it is as accurate as pathological stage classification because it does not depend on invasive procedures to obtain tissue for pathological evaluation. Breast cancer staging is improved by detecting new markers in cells, tissues or fluids that can be distinguished between different invasive stages. Advances in this field allow for faster and more reliable methods for treating breast cancer patients.

Breast cancer treatment is generally determined after an accurate staging of the primary tumor. Major treatment options include breast-conserving therapy (mastectomy, breast irradiation and axillary surgical staging) and improved radical mastectomy. Additional treatment includes termination of estrogen production by chemotherapy, regional irradiation and, in extreme cases, ovariectomy.
Until recently, the common procedure for all breast cancers was mastectomy. Fonseca et al., Annals of Internal Medicine 127: 1013 (1997). However, recent data indicate that less radical procedures can be equally effective in terms of survival for early stage breast cancer. Fisher et al., J. of Clinical Oncology 16: 441 (1998). Treatment options for patients with early stage breast cancer (ie, stage Tis) may be surgery that leaves the breast followed by local radiation therapy in the breast. Alternatively, mastectomy can optionally be used in combination with radiation or breast reconstruction. These treatment methods are equally effective in the early stages of breast cancer.

Patients with stage I and stage II breast cancer require chemotherapy and / or hormonal therapy along with surgery. Surgery has limited utility in stage III and stage IV patients. These patients are therefore good candidates for chemotherapy and radiation therapy, and surgery is limited to biopsy to allow initial stage classification or subsequent restage classification because of the disease This is because cancer is hardly curative at this stage. AJCC Cancer Staging Handbook 84, 164-65 (Irvin D. Fleming et al., Eds., 5 ^th ed. 1998).
In an effort to provide patients with more treatment options, efforts are underway to identify early-stage breast cancer that is less recurrent and can be treated with breast tumor removal without postoperative radiation treatment is there. While many trials have been made to classify early stage breast cancer, no consistent recommendations for postoperative radiation treatment are available from these studies. Page et al., Cancer 75: 1219 (1995); Fisher et al., Cancer 75: 1223 (1995); Silverstein et al., Cancer 77: 2267 (1996).

Pancreatic cancer Pancreatic cancer is the 13th most common cancer in the world and the eighth leading cause of cancer death. Donghui Li, Molecular Epidemiology, in Pancreatic Cancer 3 (Douglas B. Evans et al. Eds., 2002). In the United States, pancreatic cancer is the fourth most common cancer in both men and women, accounting for 5% of cancer deaths and nearly 30,000 deaths overall. Same as above. The proportion of pancreatic cancer is higher in men than in women and higher in African Americans than in whites. 9. Same as above. The most important predictor of pancreatic cancer is patient age; among Caucasians, the age-related incidence of pancreatic cancer is continuously increasing, as is age 85 and older. 3. Same as above. About 80% of cases occur in the age range of 60 to 80 years, and the risk of having a disease is 40 times higher in the 80s than in the 40s. Same as above. In addition, the American Cancer Society predicts 31,800 new cases of pancreatic cancer in 2004 only in the United States. Pancreatic cancer will cause about 31,200 new deaths in the same year. ACS website: cancer with the extension .org of the world wide web. Despite efforts by researchers and physicians to devise treatment for pancreatic cancer, pancreatic cancer continues to be fatal without exception. James R. Howe, Molecular Markers as a Tool for the Early Diagnosis of Pancreatic Cancer, in Pancreatic Cancer 29 (Douglas B. Evans et al. Eds., 2002).

  In addition to age, a number of risk factors for pancreatic cancer have been identified, including smoking, diet, occupation, certain medical conditions, heredity, and molecular biologic. Smoking is the most important risk factor for developing the disease, and the association between smoking and pancreatic cancer has been established by numerous studies. Li, 3 above. The relative risk is at least 1.5 and increases with the degree of smoking, reaching an outer risk ratio of 10 times. Same as above. The next most important factor appears to be diet, with increased risk associated with animal protein and fat intake and decreased risk associated with fruit and vegetable intake. 3-4 above. For certain occupations, excessive rates of pancreatic cancer are associated with chemical, coal and gas exploration, metal industry, leather tanning, textile, aluminum scouring, and transportation workers. 4. Same as above. Many medical conditions are also associated with an increased incidence of pancreatic cancer, including diabetes, chronic pancreatitis, gastrectomy, and cholecystectomy, but no causal relationship has been established between these conditions and pancreatic cancer . Same as above.

Genetic genetic factors make up less than 10% of the pancreatic cancer burden, hereditary pancreatitis, and familial cancer syndrome genes such as hMSH2 and hMLH1 (hereditary non-adenomatous colon cancer), p16 (family) Associations have been described for sex variant polymorphic mole-melanoma) and BRCA1 / BRCA2 (breast and ovarian cancer). 3 above. The hereditary basis for cancer in any organ is lower than that of the pancreas, but researchers show one or more specific genetic defects that affect an individual's susceptibility to pancreatic cancer I can't. David H. Berger & William E. Fisher, Inherited Pancreatic Cancer Syndromes, in Pancreatic Cancer 73 (Douglas B. Evans et al. Eds., 2002).

From a molecular biology standpoint, studies have revealed an association between pancreatic cancer and numerous genetic mutations, including activation of the proto-oncogene K-ras and the failure of the tumor suppressor genes p53, p16, and DPC4. Activation is included. Marina E. Jean et al., The Molecular Biology of Pancreatic Cancer, in Pancreatic Cancer 15 (Douglas B. Evans et al. Eds., 2002).

  In one study of pancreatic adenocarcinoma, 83% had K-ras activation with p16 and p53 inactivation. Same as above. K-ras mutations are found in 80-95% of pancreatic adenocarcinoma, and in pancreatic cancer, the p53, p16, and DPC4 genes are the most frequently deleted tumor suppressor genes. Howe, 29 above. Homozygote deletion, hypermethylation, and mutations in the p16 gene were found in 85-98% of pancreatic adenocarcinoma. Same as above. As expected from the role of alterations in the K-ras, p53, p16, and DPC4 genes, loss of cell cycle regulation appears to be key to pancreatic tumorigenesis, and this cancer is thus attacked May explain that it is. Jean, 15. Studies have revealed an association between this cancer and aberrant regulation of certain growth factors and growth factor receptors, and upregulation of matrix metalloproteinases and tumor angiogenesis regulators. Same as above. Epidermal growth factor, fibroblast growth factor, transforming growth factor beta, insulin-like growth factor, hepatocyte growth factor, and vascular endothelial growth factor may play a variety of roles in pancreatic cancer Is not disclosed. 18-22 above.

The development of screening techniques to detect the presence of pancreatic cancer is particularly important for this deadly cancer, which is that most patients invade the capillaries and lymph vessels that surround the pancreas. And because it does not show the presence of cancer until the tumor closes the bile duct or causes pain: Howe, supra, 29; unfortunately, patients with a translocated form of the disease are generally one year after diagnosis Less than survival, Jean et al., 15 above. Computed tomography (CT) and endoscopic retrograde cholangiopancreatography (ERCP) can assist in the diagnosis of symptomatic patients, but allow early detection at a time when treatment is possible There are currently no pancreatic tumor screening tools. Howe, 29 above. Markers such as carcinoembryonic antigen and antibodies generated against human colon cancer cell lines (CA19-9 and CA195), antibodies generated against human ovarian cancer cell lines (CA125), and humans Antibodies generated against pancreatic cancer cell lines (SPAN-1 and DUPAN-2) are elevated in the serum of patients with pancreatic cancer, but these markers appear late in the disease and due to lack of specificity. It is not enough as a reliable screening tool. Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 99 (1998); Hasholzner, U. et al., Anticancer Res. 19 (4A): 2477-80 (1999).

  Due to the current lack of appropriate screening methods, physicians are increasingly turning to techniques that use molecular biological methods as the most promising methods for early diagnosis of this disease. Howe, 30 above. Currently, there is no sensitive and highly specific marker that can detect pancreatic cancer in asymptomatic patients, but several biological markers are being investigated. Same as above. Considerable efforts are currently focused on K-ras, and researchers have devised techniques to screen pancreatic juice, bile, duodenal juice, or ERCP brushing samples to detect K-ras mutations. Since the collection of these samples is invasive and not particularly useful for screening for asymptomatic subjects, researchers are also directed to the analysis of serum and stool for K-ras mutations, the latter The former is the most promising because of the obstacles due to the complexity of the source material. 35-38, 42, same as above. Furthermore, since the serum concentration of the transcription factor protein p53 may be parallel to cancer progression, p53 has also been studied as a potential tumor marker. Ibid 37; Jean et al. Ibid 17;

Once pancreatic cancer is diagnosed, treatment decisions are made with reference to the stage of cancer progression. Numerous imaging techniques have been used for staging of pancreatic cancer, and computed tomography (CT) is the current selection method, Harmeet Kaur et al., Pancreatic Cancer: Radiologic Staging, in Pancreatic Cancer 86 (Douglas B. Evans et al. Eds., 2002); Ishiguchi, T. et al., Hepatogastroenterology 48 (40): 923-27 (2001); HJ Kim & KC Conlon, Laparascopic Staging, in Pancreatic Cancer 15 (Douglas B. Evans et al. Eds., 2002), which is frequently underestimated for the extent of cancer. MRI replaces CT at some point, especially in terms of its ability to: (1) provide contrast between various tissues, (2) modify the pulse order and modify the lesion Improve visualization and minimize artifacts, (3) perform imaging while limiting patient exposure to ionizing radiation, and (4) visualize blood vessels without using IV-iodinated contrast reagents To do. Kaur et al., 87 above. However, MRI currently does not show a clear advantage over CT. Kim & Conlon, 116 above.

Various ultrasound techniques are also currently used for stage classification, including transabdominal ultrasonography (TUS), ultrasonography (EUS), intraoperative ultrasonography (IUS), etc. EUS is one of the most promising. Kaur et al., Supra 86; Richard A. Erickson, Endoscopic Diagnosis and Staging: Endoscopic Ultrasound, Endoscopic Retrograde Cholangiopancreatography, in Pancreatic Cancer 97-106 (Douglas B. Evans et al. Eds., 2002). These techniques, however, are each limited by various factors: TUS is disturbed by gastrointestinal gas and peritoneal fat, EUS requires considerable experience in ultrasonography and endoscopy and is widely available And IUS can only be used intraoperatively. Kaur et al., 86 above.

  Although still in its early stages, the search for markers that help stage pancreatic cancer staging has found several potential clues. For example, two translocation suppressor genes, nm23-H1 and KAI1, are differentially expressed depending on the stage of pancreatic cancer, and their expression is upregulated in the early stages of the disease and downregulated in the late stages. It was found by research. Friess, H. et al., J. Clin. Oncol. 19 (9): 2422-32 (2001). Researchers are also focusing on hereditary lymph node staging, particularly searching for mutations in the K-ras proto-oncogene. Yamada, T. et al. Int’l J. Oncol. 16 (6): 1165-71 (2000). Similarly, studies have confirmed that the presence of mutated K-ras sequences in plasma / serum is associated with late stage pancreatic cancer, although the presence of early stage pancreatic cancer is also detected by this method it can. Sorenson, G.D., Clin. Cancer Res. 6 (6): 2129-37 (2000). The expected staging technique, using the reverse transcription polymerase chain reaction test with a number of markers, succeeded in identifying the stage of pancreatic cancer by analyzing blood and tissue samples for mRNA expression of the following tumor markers: : Β-human chorionic gonadotropin gene, hepatocyte growth factor receptor gene c-met, and β-1,4-N-acetyl-galactosaminyl-transferase gene. Bilchik, A. et al., Cancer 88 (5): 1037-44 (2000).

One classification system commonly used for stage classification of pancreatic cancer is the TNM system devised by Union Internationale Controlle Cancer. AJCC Cancer Staging Handbook 3 (Irvin D. Fleming et al. Eds., 5 ^th ed. 1998). This system is divided into several stages, each of which evaluates the extent of cancer growth for primary tumor (T), regional lymph nodes (N), and distant metastases (M). Same as above.
Stage 0 is characterized by carcinoma in situ (Tis), no metastasis to regional lymph nodes (N0), and no distant metastasis (M0). 113 same as above. Stages I and II differ from stage 0 only in terms of tumor category: Stage I includes tumors that are limited to the pancreas only, which are either (1) the largest dimension is 2 cm or less (T1), or (2) The largest dimension is greater than 2 cm (T2), while stage II includes a tumor (T3) that has spread directly to the duodenum, bile duct, or surrounding pancreas. Same as above. Stage III includes tumor categories T1, T2 or T3; regional lymph node translocation (N1) includes either a single lymph node (pN1a) or multiple lymph nodes (pN1b); and distant metastases No (M0). Stage IVA is characterized by a tumor that has spread directly to the stomach, spleen, colon, or adjacent large blood vessels (T4); any N category; and no distant metastases (M0). Finally, stage IVB is characterized by any T category, any N category, and distant metastasis (M1). Same as above.

Once the cancer has been staged, the only consistently effective treatment for this disease is surgery, with 10-15% of patients having a potentially curative resection Only. Jean et al., Supra, 15; Fleming et al., Eds., Supra, 111; William F. Regine, Postoperative Adjuvant Therapy: Past, Present, and Future Trial Development in Pancreatic Cancer 235 (Douglas B. Evans et al. eds., 2002). Furthermore, the 5-year survival rate of these patients undergoing resection is less than 20%. Regine, 235 above. On the other hand, chemotherapeutic agents such as gemcitabine and 5-fluorouracil have shown some efficacy against pancreatic cancer, but in reality, chemotherapy has little effect on survival from pancreatic cancer. It was. Burdette, 101 above. Although radiation therapy has shown contradictory results for its effectiveness (Id.), Radiation combined with 5-fluorouracil has shown certain promise, Regine, 235 above.

Since the prior art has failed to treat pancreatic cancer, many new approaches using molecular biology techniques are being considered. Much research has been done in the field of gene therapy, including antisense technology, gene-directed prodrug activation strategy, promoter gene strategy, and oncolytic viral therapy. Eugene A. Choi & Francis R. Spitz, Strategies for Gene Therapy, in Pancreatic Cancer 331 (Douglas B. Evans et al. Eds., 2002); Kasuya, H. et al., Hepatogastroenterology 48 (40): 957-61 (2001). Other recent approaches have focused on matrix metalloproteinase inhibition, which involves tumor cell translocation and invasion, through degradation of the basement membrane of the cells, and in peritumoral stromal degradation and angiogenesis It is an enzyme that promotes through its role. Alexander S. Rosemurgy, II & Mahmudul Haq, Role of Matrix Metalloproteinase Inhibition in the Treatment of Pancreatic Cancer, in Pancreatic Cancer 369 (Douglas B. Evans et al. Eds., 2002).

Angiogenesis in cancer
Solid tumor growth and translocation also depend on angiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman, J., 1989, Journal of the National Cancer Institute, 82, 4-6. For example, a tumor that has spread more than 2 mm must receive its own blood supply, which is achieved by inducing the growth of new capillaries. Once these new blood vessels are implanted in the tumor, they provide a means for the tumor cells to enter the circulation and translocate to a remote site, such as the liver, lung or bone. Weidner, N., et al., 1991, The New England Journal of Medicine, 324 (1), 1-8.

  Angiogenesis, defined as the growth or development of new blood vessels from existing blood vessels, is a complex process that occurs primarily during embryonic development. This process is distinguished from vasculogenesis in that new endothelial cells that cover blood vessels are created by the proliferation of existing cells rather than differentiation from stem cells. This process is invasive and relies on extracellular matrix (ECM) proteolysis, migration of new endothelial cells, and synthesis of new matrix components. Angiogenesis occurs during embryonic development of the circulatory system, but in mature humans, angiogenesis occurs only as a response to a pathological condition (except for the female reproductive cycle).

Under normal physiological conditions in adults, angiogenesis occurs only in very limited situations such as hair growth and wound healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol. Ther. 63 (3): 265-3 11; Ribatti et al., 1991, Haematologica 76 (4): 3 11-20; Risau, 1997, Nature 386 (6626): 67 1-4. Angiogenesis proceeds by a stimulus that causes the formation of a moving column of endothelial cells. Proteolytic activity is concentrated at the tip where this “vascular sprout” advances, which destroys the ECM enough to allow the column of cells to enter and migrate. Behind the advancement, endothelial cells differentiate and begin to attach to each other, forming a new basement membrane. The cells then cease to grow and eventually define a new arteriole or capillary lumen.
It has been increasingly recognized that uncontrolled angiogenesis is responsible for a wide range of diseases including, but not limited to, cancer, cardiovascular disease, rheumatoid arthritis, psoriasis and diabetic retinopathy including. Folkman, 1995, Nat Med 1 (1): 27-31; Isner, 1999, Circulation 99 (13): 1653-5; Koch, 1998, Arthritis Rheum 41 (6): 951-62; Walsh, 1999, Rheumatology ( Oxford) 28 (2): 103-12; Ware and Simons, 1997, Nat Med 3 (2): 158-64.

  Of particular importance is the observation that angiogenesis is required by solid tumors for their growth and translocation. Folkman, 1986, supra; Folkman 1990, J Natl. Cancer Inst., 82 (1) 4-6; Folkman, 1992, Semin Cancer Biol 3 (2): 65-71; Zetter, 1998, Annu Rev Med 49: 407- twenty four. Tumors usually begin as a single aberrant cell that can only grow to a size of a few cubic millimeters due to the distance from the available capillary bed, You can stay "hidden". Some tumor cells then switch to an angiogenic phenotype to activate endothelial cells, which grow and mature into new capillaries. These newly formed blood vessels not only allow continuous growth of the primary tumor, but also allow dissemination and recolonization of translocated tumor cells. Although the exact mechanisms that control angiogenesis switching are not well understood, mass neovascularization is believed to arise from a net balance between a number of pro-angiogenic and inhibitory factors. Folkman, 1995, above.

  One of the most potent angiogenesis inhibitors is endostatin identified by O'Reilly and Folkman. O'Reilly et al., 1997, Cell 88 (2): 277-85; O'Reilly et al., 1994, Cell 79 (2): 3 15-28. This discovery was based on the phenomenon that certain primary tumors can inhibit the growth of distant metastases. O'Reilly and Folkman hypothesized that the primary tumor initiates angiogenesis by producing more angiogenesis promoters than inhibitors. However, angiogenesis inhibitors reach the site of secondary tumors more than promoters because of their long half-life in the circulation. This ultimately results in primary tumor growth and secondary tumor inhibition. Endostatin is one of an increasing list of such angiogenesis inhibitors produced by primary tumors. This is a proteolytic fragment of a large protein: endostatin is a 20 kDa fragment of collagen XVIII (amino acids H1132-K1315 of mouse collagen XVIII). Endostatin has been shown to specifically inhibit endothelial cell proliferation in vitro and block angiogenesis in vivo. More importantly, administration of endostatin to tumor-bearing mice caused significant tumor regression and no toxicity or drug resistance was observed after multiple treatment cycles. Boehm et al., 1997, Nature 390 (6658): 404-407. The fact that endostatin targets genetically stable endothelial cells and inhibits various solid tumors makes endostatin a very attractive candidate for anti-cancer therapy. Fidler and Ellis, 1994, Cell 79 (2): 185-8; Gastl et al., 1997, Oncology 54 (3): 177-84; Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4: 60-3. Furthermore, angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents. Klement, 2000, J. Clin Invest, 105 (8) R15-24. Browder, 2000, Cancer Res. 6- (7) 1878-86, Arap et al., 1998, Science 279 (5349): 377-80; Mauceri et al., 1998, Nature 394 (6690): 287-91.

As noted above, each method for diagnosing and staging ovarian cancer, pancreatic cancer, lung cancer or breast cancer is limited by the technique used. Thus, there is a need for sensitive molecular and cell markers to detect ovarian cancer, pancreatic cancer, lung cancer or breast cancer. In order to optimize treatment methods, there is a need for molecular markers for accurate staging, including clinical and pathological staging of ovarian cancer, pancreatic cancer, lung cancer or breast cancer. Furthermore, there is a need for sensitive molecular and cellular markers for monitoring the progress of cancer treatment, including markers that can detect recurrence of ovarian cancer, pancreatic cancer, lung cancer or breast cancer after remission.
The present invention provides an alternative method for treating ovarian cancer, pancreatic cancer, lung cancer or breast cancer that overcomes the limitations of conventional therapies and provides additional benefits that are apparent from the detailed description below. To do.

Cellular activity of the autoimmune disease immune system is controlled by a complex network of cell surface interactions and associated signaling processes. When a cell surface receptor is activated by its ligand, a signal is transmitted to the cell, which can be inhibitory or activating depending on the signal transduction pathway involved. For many receptor systems, cellular activity is regulated by a balance between activation and inhibitory signals. In some of these, a positive signal by its ligand associated with the involvement of a cell surface receptor can be down-regulated or inhibited by a negative signal transmitted by that ligand through the involvement of a different cell surface receptor. Are known.

  The biochemical mechanisms of these positive and negative signaling pathways have been studied for a number of known immune system receptor and ligand interactions. Many receptors that mediate positive signaling have a cytoplasmic tail that includes a site of tyrosine phosphatase phosphorylation known as the immunoreceptor tyrosine-based activation motif (ITAM). A common mechanistic pathway for positive signaling involves the activation of tyrosine kinases that phosphorylate sites on the cytoplasmic domain of the receptor and on other signaling molecules. Once the receptor is phosphorylated, a binding site for the signal transduction molecule is created, which initiates the signal transduction pathway and activates the cell. The inhibitory pathway involves receptors with an immunoreceptor tyrosine-based inhibition motif (ITIM) that, like ITAM, is phosphorylated by tyrosine kinases. Receptors with these motifs are associated with inhibitory signaling, which binds to tyrosine phosphatases that block signaling by removing tyrosine from the activated receptor or signaling molecule. This is because the site is provided.

  One example of immune system activity that is regulated by a balance of positive and negative signaling is B cell proliferation. B cell antigen receptors are B cell surface immunoglobulins that when bound to an antibody mediate positive signal transduction leading to B cell proliferation. However, B cells also express FcγRIIb1, a low affinity IgG receptor. When the antigen is part of an immune complex with soluble immunoglobulins, the immune complex becomes B cell by allowing both the B cell antigen receptor and FcγRIIb1 to participate through the antigen and soluble immunoglobulin, respectively. Can be combined. The joint involvement of FcγRIIb1 and the B cell receptor complex down-regulates the activation signal and interferes with B cell proliferation. The FcγRIIb1 receptor contains an ITIM motif, which is thought to transmit an inhibitory signal to B cells through the interaction of ITIM and tyrosine phosphatase when the B cell receptor is co-involved.

  The cytolytic activity of natural killer (NK) cells is another example of immune system activity that is regulated by a balance between positive signals that initiate cell function and negative signals that interfere with activity. The receptors that activate NK cytotoxicity are not fully understood. However, if the target cell expresses a cell surface MHC class I antigen to which the NK cell has a specific receptor, the target cell is protected from being killed by NK. These specific receptors, known as killer inhibitory receptors (KIRs), when engaged by their MHC ligands, transmit negative signals and down-regulate NK cell cytotoxicity.

  KIR belongs to the immunoglobulin superfamily or the C-type lectin family (see Lanier et al., Immunology Today 17: 86-91, 1996). The known human NK KIR is a member of the immunoglobulin superfamily and exhibits differences and similarities in its extracellular, transmembrane and cytoplasmic regions. The amino acid sequence of the cytoplasmic domain common to many KIRs is an ITIM motif having the sequence YxxL / V. In some cases, phosphorylated ITIM recruits tyrosine phosphatases, which dephosphorylate molecules in the signal transduction pathway and interfere with cellular activity (Burshtyn et al., Immunity 4: 77-85, (See 1996). KIRs generally have two of these motifs separated by 26 amino acids [YxxL / V (x) .sub.26YxxL / V]. At least two NK cell receptors, each specific for the human leukocyte antigen (HLA) C allele (MHC class I molecule), exist as inhibitory and activating receptors. These receptors are very homologous in the extracellular part, but have significant differences in the transmembrane and cytoplasmic parts. One difference is the appearance of the ITIM motif in inhibitory receptors and the lack of the ITIM motif in activated receptors (Biassoni et al., Journal. Exp. Med, 183: 645-650, 1996).

  The immune receptor gp49B1 expressed by mouse mast cells is also a member of the immunoglobulin superfamily and is known to down-regulate cell activation signals and contain a pair of ITIM motifs. gp49B1 has a high degree of homology with human KIR (Katz et al., Cell Biology, 93: 10809-10814, 1996). Mouse NK cells also express a family of immune receptors, the Ly49 family, which contains the ITIM motif and functions in a manner similar to human KIR. However, the Ly49 immunoreceptor has no structural homology with human KIR and contains an extracellular C-type lectin domain, which makes it a member of the lectin superfamily of molecules (Lanier et al., Immunology Today 17 : See 86-91).

  Clearly, immune system activation and suppression signals mediated by opposing kinases and phosphatases are very important in maintaining balance in the immune system. Systems with predominant activation signals result in autoimmunity and inflammation. The immune system with a dominant suppressor signal has a low ability to challenge infected or cancer cells. Isolating new activating or inhibitory receptors is highly desirable for studying one or more biological signals that are transduced through the receptor. Furthermore, identifying such molecules provides methods for modulating and treating pathological conditions associated with autoimmunity, inflammation and infection.

  For example, involving a ligand, such as Ovr110, that interacts with a cell surface receptor having an ITIM motif with an antagonistic antibody or solubilized receptor can cause certain immune functions to be associated with suppressed immune functions. Can be used to activate in the state. On the other hand, antagonist antibodies specific for Ovr110 or solubilized forms of Ovr110 receptor are used to block the interaction between Ovr110 and cell surface receptors and in pathological conditions associated with increased immune function Specific immune function can be reduced. Conversely, a receptor lacking the ITIM motif transmits an activation signal as described above once involved, so that the effects of the antibody and solubilized receptor are reversed.

Thus, the method of diagnosing and staging autoimmune disease is limited by the technique used. Thus, there is a need for sensitive molecular and cellular markers for detecting autoimmune diseases. There is a need for molecular markers to accurately stage autoimmune diseases, including clinical and pathological staging, to optimize treatment methods. Furthermore, there is a need for sensitive molecular and cellular markers for monitoring the progress of autoimmune disease treatment, including markers that can detect recurrence of autoimmune disease after remission.
The present invention provides an alternative method for treating autoimmune diseases that overcomes the limitations of conventional therapies and provides additional benefits that will be apparent from the detailed description below.

The present invention relates to isolated Ovr110 antibodies that bind to Ovr110 on mammalian cells in vivo. The invention further relates to an isolated Ovr110 antibody that internalizes upon binding to Ovr110 on mammalian cells in vivo. The antibody may be a monoclonal antibody. Alternatively, the antibody is an antibody fragment or a chimeric or humanized antibody. The monoclonal antibody is selected from the group of hybridomas deposited under American Type Culture Collection accession numbers PTA-5180, PTA-5855, PTA-5856, PTA-5884, PTA-6266, PTA-7128 and PTA-7129 It can be produced by a hybridoma.

The antibody is a hybridoma selected from the group of hybridomas deposited under American Type Culture Collection accession numbers PTA-5180, PTA-5855, PTA-5856, PTA-5884, PTA-6266, PTA-7128 and PTA-7129. Can compete for binding to the same epitope bound by the monoclonal antibody produced by
The invention also relates to bound antibodies. They can be bound to growth inhibitors or cytotoxic agents. The cytotoxic agent can be selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes and toxins. Examples of toxins include, but are not limited to maytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin.

The mammalian cell may be a cancer cell. Preferably, the anti-Ovr110 monoclonal antibody inhibits the growth of Ovr110-expressing cancer cells in vivo.
Antibodies can be produced in bacteria. Alternatively, the antibody is produced by a hybridoma selected from the group of hybridomas having ATCC accession numbers PTA-5180, PTA-5855, PTA-5856, PTA-5884, PTA-6266, PTA-7128 and PTA-7129. Or a humanized form of the anti-Ovr110 antibody.
Preferably, the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer and breast cancer. The invention also relates to a method of producing an antibody comprising culturing appropriate cells and recovering the antibody from the cell culture.

The invention also relates to a composition comprising an antibody and a carrier. The antibody can be conjugated to a cytotoxic agent. The cytotoxic agent may be a radioisotope or other chemotherapeutic agent.
The present invention also relates to a method of killing an Ovr110-expressing cancer cell, comprising contacting the cancer cell with an antibody of the present invention, thereby killing the cancer cell. The cancer cells can be selected from the group consisting of ovarian cancer cells, pancreatic cancer cells, lung cancer cells and breast cancer cells.
The ovarian cancer or breast cancer may be ovarian serous adenocarcinoma or breast invasive ductal cancer or metastatic cancer. The breast cancer may be a HER-2 negative breast cancer. The invention also relates to a method of ameliorating an Ovr110-expressing cancer in a mammal comprising administering to the mammal a therapeutically effective amount of an antibody.

  Furthermore, the invention relates to an article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antibody as described herein. The article of manufacture can also include additional elements, eg, a package insert indicating that the composition can be used to treat ovarian cancer, pancreatic cancer, lung cancer or breast cancer.

  In addition, disorders mediated by autoimmune diseases associated with impaired signal transduction by receptors that bind to Ovr110 and down-regulate cell function may be used in patients suffering from such disorders to treat solubilized forms of Ovr110. Can be treated by administering an effective amount. Disorders mediated by pathological conditions associated with suppressed immune function can be treated by administering a therapeutically effective amount of an antagonistic Ovr110 antibody. Conversely, disorders mediated by diseases associated with impaired activation signaling by Ovr110 can be treated by administering a therapeutically effective amount of a solubilized form of Ovr110. Disorders mediated by conditions associated with autoimmune function can be treated by administering a therapeutically effective amount of an antagonistic Ovr antibody. Such autoimmune diseases include, but are not limited to: multiple sclerosis, myasthenia gravis, autoimmune neuropathies such as Guillain Valley, autoimmune uveitis, Crohn's disease, ulcerative colitis, primary Biliary cirrhosis, autoimmune hepatitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, antiphospholipid syndrome, vasculitis such as Wegener's granulomatosis, Behcet's disease, psoriasis, herpes Dermatitis, pemphigus vulgaris, vitiligo, type I or immune-mediated diabetes, Graves' disease, Hashimoto's thyroiditis, autoimmune ovitis and testicular inflammation, autoimmune diseases of the adrenal gland, rheumatoid arthritis, systemic lupus erythematosus, Scleroderma, polymyositis, dermatomyositis, spondyloarthropathy such as ankylosing spondylitis, and Sjogren's syndrome.

Detailed Description of the Invention
Definitions and General Procedures As used herein, human “Ovr110” refers to a 282 amino acid protein expressed as a glycoprotein on the cell surface, and the nucleotide and amino acid sequences of Ovr110 are, for example, WO 00/12758, cancer specific gene (CSG) Ovr110; WO 99/63088, membrane-bound protein PRO1291; WO00 / 36107, human ovarian cancer antigen; WO 02 / 02624-A2, human B7-like protein (B7-L); WO 2004/101756, OVR110; etc., the disclosures of which are expressly incorporated herein by reference. Amino acids 30-282 are presumed to be present on the cell surface. As used herein, Ovr110 also includes allelic polymorphisms and conservative substitution variants of proteins having Ovr110 biological activity.

Ovr110 is described in the literature as B7x, B7H4, B7S1, B7-H4 or B7h. Known as 5. In the NCBI RefSeq database, accession NM_024626 is noted as “T cell activation inhibitor 1 (VTCN1), homosapiens V set domain containing mRNA”. This nucleotide and the encoded protein NP_078902.1 are given the following overview:
B7H4 belongs to the B7 family of costimulatory proteins (CD80; see NIM 112203). These proteins are expressed on the surface of antigen presenting cells and interact with ligands on T lymphocytes (eg, CD28; NIM 186760). [Supplied by OMIM].

  In recent years, in a series of three independent publications, mouse and human Ovr110 are an important class of molecules that regulate T cell function activation / suppression very tightly, the costimulatory molecule T cell B7. Identified as a new member of the family. Prasad et al., B7S1, a novel B7 family member that negatively regulates T cell activation, Immunity 18: 863-73 (2003); Sica et al., B7-H4, a molecule of the B7 family, negatively regulates T cell immunity , Immunity 18: 849-61 (2003); and Zang et al., B7x: a widely expressed B7 family member that inhibits T cell activatioh, Proc. Natl Acad. Sci USA 100: 10388-92 (2003). The deduced amino acid sequence of the mouse gene for B7S1 (Prasad 2003) is very homologous to the Ovr110 molecule we identified earlier, and the deduced sequence of the human B7-H4 / B7x molecule (Sica 2003; Zang 2003) is , Ovr110. Indirect immunofluorescence analysis by flow cytometry further confirmed that our Ovr110 monoclonal antibody binds to activated T lymphocyte populations as described by these authors. A list of documents discussing Ovr110 is provided below; the disclosures of which are hereby incorporated by reference.

Our observation that Ovr110 is clearly limited to more aggressive ovarian and breast cancers is an attractive target for immunotherapy of these and potentially other types of tumors. To.
As used herein, the term “antibody” (Ab) refers to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments as long as they exhibit the desired biological activity. Including. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

  An “isolated antibody” is one that has been identified, separated and / or recovered from a component of its natural environment. Contaminated components of its natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies and can include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. Preferably, the antibodies are purified as follows: (1) antibodies determined by the Raleigh method to be greater than 95 wt% and most preferably greater than 99 wt% (2) rotating cup sequencing Reduced to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using the apparatus, or (3) by SDS-PAGE using Coomassie blue or preferably silver staining Alternatively, purify it uniformly under non-reducing conditions. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (the IgM antibody is called the J chain) Consisting of 5 basic heterotetrameric units with additional polypeptides, thus containing 10 antigen binding sites, while secreted IgA antibodies polymerize to 2-5 basic with J chain A multivalent assembly comprising four chain units of In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. . Each H and L chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has, at the N-terminus, a variable region (VH) followed by three constant regions (CH) for each of the α and γ chains and four CH regions for the L and F isotypes. Each 6L chain has a variable region (VL) at the N-terminus followed by a constant region (CL) at the other end.

VL aligns with VH and CL aligns with the first constant region (CHI) of the heavy chain.
Certain amino acid residues are believed to form an interface between the light chain variable region and the heavy chain variable region. The pairing of VH and VL together forms a single antigen binding site. The structure and properties of the different classes of antibodies, ^{e.g., Basic and Clinical Immunology, 8 th} edition, Daniel P. Stites, Abba I. Teff and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994 , Page 71 and chapter 6.

Based on the amino acid sequences of these constant regions, light chains from all vertebrate species can be assigned to one of two clearly distinguishable classes called kappa and lambda. Depending on the amino acid sequence of the constant region of these heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains denoted α, δ, ε, γ and μ, respectively. γ and α classes are further divided into subclasses on the basis of relatively non major differences in _{C H} sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4 , IgA1 and IgA2.

The term “variable” refers to the fact that certain segments of the variable region vary greatly between antibodies with respect to sequence. The V region mediates antigen binding and defines the specificity of a particular antibody for this particular antigen. However, variability is not evenly distributed over the 1-10 amino acid range of the variable region. Instead, the V region is a 15-30 amino acid framework region separated by very highly variable relatively short regions called “hypervariable regions”, each 9-12 amino acids in length. It consists of a relatively invariant extension called (FR). The natural heavy and light chain variable regions each contain four FRs, which take a large P-sheet shape and are joined by three hypervariable regions that form a loop to form a P-sheet structure. And in some cases forms part of this. The hypervariable regions in each chain are held closely together by FR and together with the hypervariable region from the other chain contribute to the formation of the antigen binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest , see ^{5 th Ed. Public Health Service,} National Institutes of Health, Bethesda, MD. (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits involvement in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues involved in antigen binding of an antibody. The hypervariable region generally, "complementarity determining region" or "CDR" (e.g., approximate residues 24-34 (L1 in VL), 5 0~5 6 (L2 ) and 89-97 (L3), And approximately 1-35 (H1), 50-65 (H2) and 95-102 (113) in VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Amino acid residues from Bethesda, MD. (1991)) and / or “hypervariable loops” (eg residues 26-32 (L1), 50-52 (L2) and 91-96 (U) in VL, And these residues from 26-32 (H1), 53-55 (1-12) and 96-101 (H3) in VH; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)) including.

  As used herein, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies that make up the population can be naturally occurring, which can be present in small amounts. Identical except for sexual mutants. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without being contaminated by other antibodies. The modifier “monoclonal” should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or bacterial, eukaryotic animal or plant cells. Can be produced using recombinant DNA methods in US Pat. No. 4,816,567. “Monoclonal antibodies” can also be obtained from phage antibody libraries, for example in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Isolation can be done using the techniques described.

  Monoclonal antibodies herein include “chimeric” antibodies and fragments thereof as long as they exhibit the desired biological activity, which is a portion of its heavy and / or light chain. Is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining one or more chains are The same or homologous to the corresponding sequence in antibodies from other species or belonging to other antibody classes or subclasses (US Pat. No. 4,816,567; and Morrison et al.) al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Related chimeric antibodies are herein referred to as “primatized” comprising variable region antigen binding sequences and human constant region sequences derived from non-human primates (eg, Old World monkeys, apes, etc.). Including antibodies.

  An “intact” antibody is one that includes an antigen binding site and CL and at least a heavy chain constant region, CHI, CH2 and CH3. The constant region may be a native sequence constant region (eg, a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

  “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2 and Fv fragments; diabody; linear antibodies (US Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); single chain antibody molecules; as well as multi-selective antibodies formed from antibody fragments. Papain digestion of the antibody yields two identical antigen-binding fragments called the “Fab” fragment and the remaining “Fc” fragment, the display reflecting the ability to easily crystallize. The Fab fragment consists of a complete light chain with the variable region domain of the heavy chain (VH) and the first constant region (CHI) of one heavy chain. Each Fab fragment is monovalent for antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody yields one large F (ab ′) 2 fragment, which roughly corresponds to two Fab fragments with disulfide binding, has bivalent antigen binding activity, and still crosslinks antigen be able to. Fab 'fragments differ from Fab fragments by having several additional residues at the carboxy terminus of the CHI region including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab 'in which one or more cysteine residues in the constant region have a free thiol group. F (ab ') 2 antibody fragments originally were produced as pairs of 8 Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains a carboxy terminal portion where both heavy chains are held together by disulfides. Antibody effector functions are determined by sequences in the Fc region, which is also the portion recognized by the Fc receptor (FcR) found on certain types of cells.
“Fv” is the minimum antibody fragment which contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain in a strict, non-covalent association. From the folding of these two regions, 6 hypervariable loops (3 loops each from the heavy and light chains) radiate, which contributes to amino acid residues for antigen binding and antigen binding specificity to the antibody. Is granted. However, even a single variable region (or half of the Fv containing only three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but not more than the complete binding site At low affinity.

  “Single-chain Fv” is also abbreviated as “sFv” or “scFv”, which is an antibody fragment comprising VH and VL antibody regions bound to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL regions so that the sFv can form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.

  The term “diabody” is a small antibody fragment such that pairing is achieved between V region chains rather than within the chain, resulting in a bivalent fragment, ie a fragment having two antigen binding sites. Means the small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) with a short linker (about 5-10 residues) between the VH and VL regions. Bispecific diabodies are heterodimers of two “crossover” sFv fragments, where the VH and VL regions of the two antibodies are on different polypeptide chains. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

A “native sequence” polypeptide is one having the same amino acid sequence as a polypeptide (eg, antibody) derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring human polypeptide, mouse polypeptide or polypeptide from any other mammalian species.
The term “amino acid sequence variant” means a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Typically, an amino acid sequence variant of Ovr110 has at least about 70% homology with native sequence Ovr110, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90% homology, And most preferably has at least about 95% homology. Amino acid sequence variants may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence.

The phrase “functional fragment or analog” of an antibody is a compound that has qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that can bind to IgE immunoglobulin, wherein the ability of such a molecule to bind to the high affinity receptor, FcεRI. Those that can be combined in a manner that prevents or substantially reduces.
“Homology” is defined as the percentage of residues in an amino acid sequence variant that are identical after aligning the sequences and optionally introducing gaps to achieve the greatest percentage of homology. . Methods and computer programs for alignment are well known in the art. Sequence similarity can be measured by any common sequence analysis algorithm, such as GAP or BESTFIT or other variation Smith-Waterman alignment. See TF Smith and MS Waterman, J. Mol. Biol. 147: 195-197 (1981) and WR Pearson, Genomics 11: 635-650 (1991).

  “Humanized” forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are non-human species (donor antibodies) in which residues from the recipient hypervariable region have the desired antibody specificity, affinity and ability, eg, mouse, rat, rabbit or A human immunoglobulin (receptor antibody) that is replaced by a non-human primate residue from a hypervariable region. In some examples, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody comprises at least one, typically substantially all of the two variable regions, wherein all or substantially all of the hypervariable loops correspond to that of a non-human immunoglobulin. However, all or substantially all of the FR is of human immunoglobulin sequence. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593- See 596 (1992).

  As used herein, an “internalized” anti-Ovr110 antibody is one that is taken up (ie, entered) by a cell by binding to Ovr110 (ie, cell surface Ovr110) on a mammalian cell. Internalized antibodies naturally include antibody fragments, human or humanized antibodies and antibody conjugates. For therapeutic use, in vivo internalization is intended. The number of internalized antibody molecules is sufficient or appropriate to kill Ovr110-expressing cells, particularly Ovr110-expressing cancer cells. Depending on the efficacy of the antibody or antibody conjugate, in some instances, the uptake of one antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly effective at killing, such that internalization of one molecule of toxin bound to an antibody is sufficient to kill tumor cells.

  Whether an anti-Ovr110 antibody is internalized upon binding to Ovr110 on a mammalian cell can be determined by various assays, including those described in the following experimental examples. For example, to test internalization in vivo, a test antibody is labeled and introduced into an animal known to have Ovr110 expressed on the surface of certain cells. The antibody can be radiolabeled or labeled with, for example, fluorescent or gold particles. Suitable animals for this assay include mammals such as NCR nude mice, including human Ovr110 expressing tumor transplants or xenografts, or mice transfected with cells transfected with human Ovr110, or transgenics that express the human Ovr110 transgene. Includes a mouse. Appropriate controls are given animals that have not been given or not associated with the test antibody, and antibodies to other antigens on the relevant cells, and this antibody binds to the antigen. Includes animals known to internalize. The antibody can be administered to the animal, for example, by intravenous injection. At suitable time intervals, tissue sections of this animal were prepared using known methods or as described in the following experimental examples, internalized and internalized in cells by light or electron microscopy. The position of the antibody can be analyzed. For in vitro internalization, cells can be incubated in tissue culture dishes in the presence or absence of relevant antibodies added to the culture medium and processed for microscopic analysis at the desired time point.

  The presence of internalized labeled antibody in the cells can be visualized directly by microscopy or, if radiolabeled antibodies are used, by autoradiography. Alternatively, in a quantitative biochemical assay, a population of cells containing Ovr110-expressing cells is contacted in vitro or in vivo with a radiolabeled test antibody and cells (if contacted in vivo). The cells are then isolated after a suitable amount of time), or treated with protease or acid washed to remove non-internalized antibodies on the cell surface. The cells are crushed and the protease resistance radiometric value per minute (cpm) associated with each batch of cells is measured by passing the homogenate through a scintillation counter. Based on the known specific activity of the radiolabeled antibody, the number of antibody molecules internalized per cell can be estimated from the scintillation counts of the crushed cells. The cells are obtained in vitro, preferably in solution form, for example, by adding the cells to the cell culture medium in a culture dish or flask and mixing the antibody thoroughly with the medium to homogenize the cells against the antibody. “Contact” with the antibody by ensuring proper exposure. Instead of adding to the culture medium, the cells can be contacted with the test antibody in an isotonic solution such as PBS in a test tube for a desired time. When administered to a patient, the cells are contacted with the antibody in vivo by any suitable method of administering the test antibody, eg, the administration method described below.

  As the rate of internalization of antibodies upon binding to Ovr110-expressing cells in vivo increases, the desired killing or growth inhibitory effect on the target Ovr110-expressing cells is achieved more quickly, for example, with cytotoxic immunoconjugates Can do. Preferably, the internalization kinetics of anti-Ovr110 antibodies are such that they prefer rapid killing of Ovr110-expressing target cells. Accordingly, the anti-Ovr110 antibody is preferably within 24 hours after administration of the antibody in vivo, more preferably within about 12 hours, even more preferably within about 30 minutes to 1 hour, most preferably about 30 It is desirable to show a rapid rate of internalization within minutes. The present invention provides an antibody that rapidly internalizes in about 15 minutes after the introduction of the anti-Ovr110 antibody in vivo. The antibody is preferably internalized into the cell within a few hours, preferably within an hour, even more preferably within 15-30 minutes upon binding to Ovr110 on the cell surface.

  To test whether a test antibody can compete for binding to the same epitope bound by an anti-Ovr110 antibody of the present invention, including an antibody produced by a hybridoma deposited with the ATCC Alternatively, a cross-blocking assay, such as a competitive ELISA assay, can be performed. In an exemplary competitive ELISA assay, microtiter plate Ovr110 coated wells or Ovr110 coated sepharose beads are preincubated with or without candidate competitive antibodies and then biotinylated books. Add anti-Ovr110 antibody of the invention. The amount of labeled anti-Ovr110 antibody bound to the Ovr110 antigen in the wells or on the beads is measured using an avidin-peroxidase conjugate and an appropriate substrate.

  Alternatively, the anti-Ovr110 antibody can be labeled with, for example, a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled anti-Ovr110 antibody that binds to the antigen has an inverse correlation to the ability of the candidate competitive antibody (test antibody) to compete for binding to the same epitope on the antigen. That is, as the affinity of the test antibody for the same epitope increases, a smaller amount of labeled anti-Ovr110 antibody binds to the well coated with antigen. Compared to a control in which the candidate competitive antibody is run in parallel in the absence of the candidate competitive antibody (but may be in the presence of a known non-competitive antibody), the binding of the anti-Ovr110 antibody If the candidate competitive antibody is capable of blocking at least 20%, preferably at least 20-50%, and even more preferably at least 50%, the candidate competitive antibody is substantially at the same epitope as the anti-Ovr110 antibody of the invention. An antibody that binds or is considered to be an antibody that competes for binding to the same epitope. It will be appreciated that modifications of this assay can be made to reach the same quantitative numerical data.

  Antibodies having the “biological properties” of the specified antibody, such as the following monoclonal antibodies: Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1 Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110. C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1 , Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110 .I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15 , Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3 Have one or more of the biological characteristics of the designated antibody to distinguish it from the antibody Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77 .1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5. 1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110 .C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110 .I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15, Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21 , Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3 are Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110. A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110 .C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16. 1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15, Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110. It binds to the same epitope as that bound by J3 (for example, the following monoclonal antibodies: Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1 , Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110. I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6, Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15, Ovr110.I16, Ovr110. I17, Ovr110.I18, Ovr110.I20, Ovr110.I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3 compete with the binding to Ovr110 or block the binding to Ovr110), in Ovr110-expressing tumor cells can be targeted in vivo and can be internalized upon binding to Ovr110 on mammalian cells in vivo. Similarly, Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110 .A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110. C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1 , Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6 , Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15, Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110 Antibodies with the biological properties of the I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3 antibodies have the same epitope binding, targeting, internalization, tumor growth inhibition and cytotoxicity as the antibodies described above. Has characteristics.

  The term “antagonist” antibody is used in the broadest sense and includes antibodies that partially, or completely block, inhibit or neutralize the biological activity of the native Ovr110 protein disclosed herein. A method for identifying an antagonist of an Ovr110 polypeptide comprises contacting an Ovr110 polypeptide or a cell that expresses Ovr110 on the cell surface with a candidate antagonist antibody, and 1 or 2 normally associated with the Ovr110 polypeptide. Measuring a detectable change in the above biological activity can be included.

  An “antibody that inhibits the growth of tumor cells that express Ovr110” or “growth-inhibitory” antibodies are those that bind to cancer cells that express or overexpress Ovr110, resulting in measurable growth inhibition. Preferred growth inhibitory anti-Ovr110 antibodies have a growth of Ovr110 expressing tumor cells (eg, ovarian cancer cells, pancreatic cancer cells, lung cancer cells and breast cancer cells) greater than 20%, preferably from about 20% to Inhibits about 50%, and even more preferably greater than 50% (eg, about 50% to about 100%), and the control is typically tumor cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1-30 pg / ml or about 0.5 nM-200 nM in cell culture, where growth inhibition 1- Determined after 10 days. In vivo inhibition of tumor cell growth can be determined in a variety of ways, as described in the Experimental Examples section below. By administering the anti-Ovr110 antibody at about 1 pg / kg body weight to about 100 mg / kg body weight, the decrease in tumor size or tumor cell proliferation is preferably within about 5 days to 3 months from the first administration of the antibody, preferably An antibody is growth inhibitory in vivo if it occurs within about 5-30 days.

  An “apoptosis-inducing” antibody is determined by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation and / or membrane vesicle formation (called apoptotic bodies), a program Induced cell death. The cell is usually one that overexpresses Ovr110. Preferably, the cell is a tumor cell, such as an ovarian cell, pancreatic cell, lung cell or breast cell. A variety of methods are available to assess cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering; and nuclear / chromatin condensation associated with DNA fragmentation is reduced It can be assessed by any increase in diploid cells. Preferably, an antibody that induces apoptosis results in an induction of annexin binding in annexin binding assays that is about 2-50 times, preferably about 5-50 times, most preferably about 10-50 times that of untreated cells. Is.

  Antibody “effector function” refers to the biological activity attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( Downregulation of eg B cell receptors); and B cell activation.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” binds to Fc receptors (FcR) present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils and macrophages, A cytotoxic form in which the secreted Ig allows these cytotoxic effector cells to specifically bind to the target cell with the antigen and then kill the target cell with the cytotoxic agent. means. Antibodies “arm” cytotoxic cells and are absolutely necessary for such killing. NK cells, the main cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of the relevant molecule, an in vitro ADCC assay, such as the assay described in US Pat. No. 5,500,362 or 5,821,337, can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the relevant molecule is evaluated in vivo, for example in an animal model as disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). Can do.

  “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII and FcγRIII subclasses, and allelic polymorphisms and alternatively spliced forms of these receptors. Including. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See review M. of Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126.330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, associated with the transport of maternal IgG into the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cell expresses at least FcγRIII and exhibits ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMC and NK cells are preferred. Effector cells can be isolated from natural sources, such as blood.
“Complement dependent cytotoxicity” or “CDC” means lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind to these cognate antigens. To assess complement activation, the CDC assay described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed.

  The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancy. More specific examples of such cancers include squamous cell carcinoma (eg epithelial squamous cell carcinoma) and lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, peritoneal Cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer , Colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, spawning cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, black Tumors, multiple myeloma and B-cell lymphoma, brain and head and neck cancer, and related metastases.

  An “Ovr110 expressing cell” is a cell that expresses endogenous or transfected Ovr110 on the cell surface. An “Ovr110-expressing cancer” is a cancer that includes cells having Ovr110 protein present on the cell surface. An “Ovr110-expressing cancer” produces Ovr110 on the surface of this cell at a level sufficient to allow an anti-Ovr110 antibody to bind and have a therapeutic effect on the cancer. A cancer that “overexpresses” Ovr110 has a significantly higher level of Ovr110 at the cell surface compared to non-cancerous cells of the same tissue type. Such overexpression can occur by gene amplification or by increased transcription or translation. Ovr110 overexpression can be determined by assessing increased levels of Ovr110 protein present on the surface of cells in a diagnostic or prognostic assay (eg, by immunohistochemical assay; FACS analysis). Alternatively or additionally, the level of nucleic acid or mRNA encoded by Ovr110 in the cell can be measured, for example by fluorescence in situ hybridization; (FISH; see WO98 / 45479 published October 1998), Southern blotting , Northern blotting or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR). Ovr110 overexpression can also be examined by measuring antigen in biological fluids such as serum using, for example, antibody-based assays (see, eg, US Pat. No. 4,933,294, June 1990). Published 12 days; W091 / 05264, published 18 April 1991; US Pat. No. 5,401,638, published 28 March 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990). ). Apart from the assays described above, various in vivo assays are available to skilled practitioners. For example, cells in a patient's body can be exposed to an optionally detectable label, eg, an antibody labeled with a radioisotope, and binding of this antibody to the patient's cells is externally scanned, eg, for radioactivity. Or by analyzing a biopsy taken from a patient previously exposed to the antibody. The Ovr110 expressing cancer includes ovarian cancer, pancreatic cancer, lung cancer or breast cancer. Body fluid includes all bodily fluids, secretions, released and derived fluids of the body, such as blood, plasma, serum, urine, saliva, sputum, tears, ascites, peritoneal lavage fluid, lymph fluid, bile, semen, Pus, amniotic fluid, aqueous humor, earwax, milk cake, sputum, intestinal fluid, menstruation, milk, mucus, pleural fluid, sweat, vaginal lubricating fluid, vomiting, cerebrospinal fluid and synovial fluid.

  “Mammal” for the purpose of treating cancer or ameliorating symptoms of cancer means any mammal, humans, domestic and farm animals, as well as zoos, sports or pet animals, such as Includes dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like. Preferably the mammal is a human.

  “Treat” or “treatment” or “remission” means both therapeutic treatment and prophylactic or preventative measures, wherein the goal is to prevent or prevent the targeted pathological condition or disorder To delay (reduce). Those in need of treatment include those that already have a disorder and those that tend to have a disorder or that should be prevented. The subject or mammal has received one or two of the following observable and / or measurable reductions or lack of this after a therapeutic amount of anti-Ovr110 antibody has been administered by the methods of the invention: Where indicated, a cancer cell that has been successfully “treated” for an Ovr110-expressing cancer: a reduction in the number of cancer cells or a lack of cancer cells; a reduction in tumor size; Inhibition of tumor invasion into peripheral organs (ie, delayed to some extent and preferably stopped); inhibition of tumor metastasis (ie, delayed to some extent and preferably stopped); inhibition to some extent of tumor growth; and / or Remission to some extent of one or more symptoms associated with a particular cancer; reduction in morbidity and mortality, and improvement in quality of life issues. To the extent that an anti-Ovr110 antibody can prevent the growth of existing cancer cells and / or kill it, it can be cytostatic and / or cytotoxic. Reduction of these signs or symptoms can also be perceived by the patient.

The above parameters for assessing successful treatment and improvement in the disease can be readily measured by routine procedures well known to physicians. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).
The term “therapeutically effective amount” means an amount of an antibody or agent effective to “treat” a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into peripheral organs (ie, is delayed to some extent and preferably Tumor metastasis is inhibited (ie delayed and preferably stopped to some extent); tumor growth is inhibited to some extent; and / or one or more of the symptoms associated with cancer are to some extent Can be in remission. See the above definition of “treat”. To the extent that the agent can prevent the growth of existing cancer cells and / or kill it, it can be cytostatic and / or cytotoxic.

“Chronic” administration allows one or more agents to be administered in a continuous fashion relative to the acute regime so that the initial therapeutic effect (activity) is maintained over an extended period of time. Means that.
“Intermittent” administration is not a continuous, uninterrupted, but rather periodic treatment.
Administration “in combination with” one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.
As used herein, “carrier” includes pharmaceutically acceptable carriers, additives or stabilizers that are non-toxic to the cells or mammals exposed at the dosages and concentrations employed.

  Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins For example serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; Sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS® Including It is.

The term “cytotoxic agent” as used herein means a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioisotopes (eg, radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , and Lu), chemotherapeutic agents such as methotrexate. (methotrexate), adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, Chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleases, antibiotics and toxins such as small molecule toxins or enzymatically active of bacterial, fungal, plant or animal origin A toxin, including these fragments and / or variants , For example, gelonin (gelonin), lysine is intended to include the various antitumor or anticancer agents disclosed saporin and below. Other cytotoxic agents are described below. Tumoricides cause destruction of tumor cells.

  As used herein, “growth inhibitor” means a compound or composition that inhibits the growth of cells, particularly Ovr110-expressing cancer cells, either in vitro or in vivo. Thus, a growth inhibitor may be one that significantly reduces the percentage of Ovr110 expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce GI arrest and M-phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Agents that stop GI also affect S-phase arrest, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and alla. (ara) -C. For more information, see The Molecular Basis of Cancer, Mendelsohn and Israel edited by Murakami et al., Chapter 1, “Cell Cycle Regulation, Oncogenes and Antineoplastic Drugs” (WB Saunders: Philadelphia, 1995), especially page 13. Can be found inside. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from yew. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer) derived from European yew is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel facilitate the assembly of microtubules from tubulin dimers and stabilize the microtubules by preventing depolymerization, thereby resulting in inhibition of mitosis in the cell.

  As used herein, “label” refers to a detectable compound or composition that binds directly or indirectly to an antibody to yield a “labeled” antibody. The label may itself be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, catalyzes a chemical change in the detectable substrate compound or composition. Also good.

  As used herein, the term “epitope tagged” refers to a chimeric polypeptide comprising an anti-Ovr110 antibody polypeptide fused to a “tag polypeptide”. The tag polypeptide has residues that are sufficient to provide an epitope from which an antibody can be made, but short enough that it does not interfere with the activity of the fused Ig polypeptide. The tag polypeptide is also preferably very unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually 8-50 amino acid residues (preferably about 10-20 amino acid residues).

“Small molecule” is defined herein as having a molecular weight of less than about 500 Daltons.
The term “package insert” is customarily included in the commercial packaging of therapeutic products and includes instructions for use, use, dosage, administration, contraindications and / or precautions regarding the use of such therapeutic products. Used to mean book.
An “isolated nucleic acid molecule” is a nucleic acid molecule, such as RNA, DNA, or a mixed polymer, which is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, necessarily With natural sequences. The term encompasses nucleic acid molecules removed from this naturally occurring environment and is biologically synthesized by recombinant or cloned DNA isolates and chemically synthesized analogs or heterologous systems. Containing analogs. A substantially pure nucleic acid molecule includes an isolated form of the nucleic acid molecule.

“Vector” includes shuttle vectors and expression vectors, including, for example, plasmids, cosmids or phagemids. Typically, a plasmid construct also includes an origin of replication (eg, a ColEl origin of replication) and a selectable marker (eg, ampicillin or tetracycline resistance), respectively, for plasmid replication and selection in bacteria. “Expression vector” means a vector containing control sequences or regulatory elements required for the expression of an antibody, including an antibody fragment of the invention, in prokaryotes, eg, bacteria or eukaryotic cells. Suitable vectors are disclosed below.
Cells that produce the anti-Ovr110 antibodies of the present invention include parental hybridoma cells, eg, hybridomas deposited with the ATCC, and bacterial and eukaryotic host cells into which the nucleic acid encoding the antibody has been introduced. Suitable host cells are disclosed below.

  RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNA (siRNA) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is generally referred to as post-transcriptional gene silencing or RNA silencing, and also means arrest in fungi. The post-transcriptional gene silencing process is thought to be an evolutionarily conserved cytoprotective mechanism used to prevent the expression of foreign genes, commonly shared by various flora and gates (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression stems from viral infection or random integration of transposon elements into the host genome by cellular responses that specifically destroy homologous single-stranded RNA or viral genomic RNA It may have evolved in response to the production of double stranded RNA (dsRNA). Although the presence of dsRNA in the cell triggers an RNAi response, the mechanism has not yet been fully characterized. This mechanism results from dsRNA-mediated activation of protein kinases PKR and 2 ', 5'-oligoadenylate synthetase, resulting in an interferon response that results in nonspecific cleavage of mRNA by ribonuclease L it is conceivable that.

  The presence of long dsRNA in the cell stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer is involved in processing dsRNA into short pieces of dsRNA, known as small interfering RNA (siRNA) (Berstein et al., 2001, Nature, 409, 363). Small interfering RNAs derived from Dicer activity are typically about 21-23 nucleotides in length and contain about 19 base pair duplexes. Dicer was also involved in the excision of 21 and 22 nucleotide small transient RNAs (stRNAs) from conserved RNA precursors involved in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also involves an endonuclease complex containing siRNA, commonly referred to as RNA-induced silencing complex (RISC), that mediates the cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Features. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

  RNAi mediated by small interfering RNA has been studied in various systems. Fire et al., 1998, Nature, 391, 806 first observed RNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describes RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293 describes RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 describes RNAi induced by double-stranded introduction of synthetic 21 nucleotide RNA in cultured mammalian cells including human embryonic kidney and HeLa cells. . Recent studies in Drosophila embryo lysates (Elbashir et al., 2001, EMBO J., 20, 6877) have shown that siRNA length, structure, chemical composition and sequence essential to mediate efficient RNAi activity Certain requirements for became clear. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3'-overhangs. In addition, complete replacement of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas deoxy of 3'-terminal siRNA overhanging nucleotides. Substitution with nucleotide (2'-H) has been shown to be tolerated. A single mismatch sequence at the center of the siRNA duplex was also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5 ′ end rather than the 3 ′ end of the siRNA guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies indicate that 5 'phosphate on the target complementary strand of the siRNA duplex is required for siRNA activity and that ATP maintains the 5' phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

  Studies have shown that replacing the 3 'overhang segment of a 21-mer siRNA duplex with two nucleotide 3' overhangs with deoxyribonucleotides has no adverse effect on RNAi activity. It was. It has been reported that substitution of up to 4 nucleotides on each end of siRNA with deoxyribonucleotides is well tolerated, while complete substitution with deoxyribonucleotides does not result in RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition, Elbashir et al., Supra, also report that replacement of siRNA with 2'-O-methyl nucleotides completely abolishes RNAi activity. Both Li et al., International PCT Publication WO 00/44914 and Beach et al., International PCT Publication WO 01/68836, both indicate that siRNAs contain modifications to either the phosphate-sugar backbone or the nucleoside. Suggesting that it can contain and contain at least one nitrogen or sulfur heteroatom, but neither application teaches to what extent these modifications are tolerated in siRNA molecules, No examples of modified siRNA are provided. Kreutzer and Limmer, Canadian Patent Application No. 2,359,180, is also used in dsRNA constructs for double-stranded RNA-dependent protein kinase PKR, particularly 2'-amino or 2'-O-methyl nucleotides, and 2'- Certain chemical modifications have been described to prevent activation of nucleotides containing O or 4'-C methylene bridges. However, Kreutzer and Limmer similarly do not show to what extent these modifications are tolerated in siRNA molecules and do not provide any examples of such modified siRNAs.

  Parrish et al., 2000, Molecular Cell, 6, 1977-1087 tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (> 25 nt) siRNA transcripts. The authors described the introduction of thiophosphate residues into these siRNA transcripts by including thiophosphate nucleotide analogs with T7 and T3 RNA polymerases, and “two (phosphorothioate) modified bases”. RNA also has a significant decrease in effectiveness as an RNAi trigger (data not shown); modification of more than two residues (phosphorothioate) greatly destabilizes RNA in vitro, We observed that we were unable to analyze the interference activity. 1081 same as above. The author also tested certain modifications at the 2 'position of the nucleotide sugar in long siRNA transcripts and observed that substitution of deoxynucleotides with ribonucleotides "remarkably reduced interference activity", especially This was the case in the case of substitution of uridine for thymidine and / or cytidine for deoxycytidine. Same as above. In addition, this author includes 4-thiouracil, 5-bromouracil, 5-iodouracil, 3- (aminoallyl) uracil substitution with uracil, and inosine with guanosine in the sense and antisense strands of siRNA. Tested for certain base modifications, when included in any chain, 4-thiouracil and 5-bromouracil were all well tolerated, while inosine "remarkably reduced interference activity" I found it. Introduction of 5-iodouracil and 3- (aminoallyl) uracil in the antisense strand also resulted in a significant decrease in RNAi activity.

  Beach et al., International PCT Publication WO 01/68836, describes a specific method of attenuating gene expression using endogenously derived dsRNA. Tuschl et al., International PCT Publication WO 01/75164 describes the in vitro Drosophila RNAi system and the use of certain siRNA molecules for certain functional genomes and certain therapeutic applications; Tuschl, 2001, Chem. Biochem., 2, 239-245 suggests that RNAi can not be used to treat genetic diseases or viral infections because of the "risk of activating the interferon response". Be suspicious of. Li et al., International PCT Publication WO 00/44914, describes the use of specific dsRNAs for use in attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication WO 01/36646, describes a method for inhibiting the expression of certain genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication WO 99/32619 describes a specific method for introducing certain dsRNA molecules into cells for use in inhibiting gene expression. Plaetinck et al., International PCT Publication WO 00/01846 describes a method for identifying specific genes responsible for conferring specific phenotypes in cells using specific dsRNA molecules. Yes. Mello et al., International PCT Publication WO 01/29058 describes the identification of specific genes involved in dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCT Publication WO 99/07409, describes specific compositions of specific dsRNA molecules combined with certain antiviral agents. Driscoll et al., International PCT Publication WO 01/49844 describes specific DNA constructs for use in promoting gene silencing in target organisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087 describes specific chemically modified siRNA constructs that target the C. elegans unc-22 gene. Tuschl et al., International PCT Publication WO 02/44321, describes certain synthetic siRNA constructs.

Compositions and Methods of the Invention The present invention provides anti-Ovr110 antibodies. Preferably, the anti-Ovr110 antibody is internalized upon binding to cell surface Ovr110 on mammalian cells. Anti-Ovr110 antibodies can also destroy or cause this destruction of tumor cells with Ovr110.

  It was not clear that Ovr110 was competitive with internalization. Furthermore, the ability of an antibody to internalize depends on several factors, including affinity, binding activity, and antibody isotype, and the epitope to which it binds. The inventors herein have demonstrated that cell surface Ovr110 is internalization competitive when bound by an anti-Ovr110 antibody of the present invention. Furthermore, it has been demonstrated that the anti-Ovr110 antibodies of the present invention can specifically target Ovr110-expressing tumor cells in vivo and inhibit or kill these cells. Due to their in vivo tumor targeting, internalization and growth inhibitory properties of anti-Ovr110 antibodies, these antibodies are in therapeutic use, for example in the treatment of various cancers including ovarian cancer, pancreatic cancer, lung cancer or breast cancer. , Very suitable. Internalization of an anti-Ovr110 antibody is, for example, when the antibody or antibody conjugate has an intracellular site of action and when a cytotoxic agent (eg, toxin calicheamicin) bound to this antibody does not easily cross the plasma membrane It is preferable. Internalization is not necessary if the antibody or agent bound to it does not have an intracellular site of action, for example if the antibody can kill tumor cells by ADCC or some other mechanism.

  The anti-Ovr110 antibodies of the present invention also have various non-therapeutic uses. The anti-Ovr110 antibodies of the invention can be useful for diagnosis and staging of Ovr110 expressing cancer (eg, in radioimaging). These can be used alone or in combination with other ovarian cancer markers, including but not limited to CA125, HE4 and mesothelin. This antibody can also be used as a step in the purification of other cells to purify or immunoprecipitate Ovr110 from cells, eg, to detect and quantify Ovr110 in vitro, such as in an ELISA or Western blot. Is useful for killing and excluding from a population of mixed cells. The internalizing anti-Ovr110 antibody of the present invention may be in various forms encompassed by the definition of “antibody” herein. Thus, antibodies include full-length or intact antibodies, antibody fragments, native sequence antibodies or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates and functional fragments thereof. In a fusion antibody, the antibody sequence is fused to a heterologous polypeptide sequence. The antibody can be modified in the Fc region to provide the desired effector function. As described in more detail in the following sections, naked antibodies bound on the cell surface using appropriate Fc regions can be obtained, for example, by antibody-dependent cytotoxicity (ADCC) or by complement-dependent cytotoxicity. Cytotoxicity can be induced by acquiring complement in action or by some other mechanism. Alternatively, certain other Fc regions can be used where it is desirable to eliminate or reduce effector function to minimize side effects or therapeutic complications.

  The antibody can compete for or bind to substantially the same epitope bound by the antibody of the invention. Antibodies with the biological characteristics of this anti-Ovr110 antibody of the present invention are also contemplated, including, for example, ATCC accession number PTA-, including in vivo tumor targeting, internalization and all cell growth inhibition or cytotoxic characteristics. Anti-Ovr110 antibody with the biological characteristics of monoclonal antibodies produced by hybridomas consistent with 5180, PTA-5855, PTA-5856, PTA-5884, PTA-6266, PTA-7128 and PTA-7129. Specifically provided are amino acids 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120 of human Ovr110. -130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250 250-260, 260-270, 270-282, or 21-35, 31-45, 41-55, 51-65, 61-75, 71-85, 81-95, 91-105, 101-115, 111-125, 121-135, 131-145, 141-155, 151-165, 161-175, 171-185, 181-1 It binds to an epitope present in 5,191～205,201～215,211～225,221～235,231～245,241～255,251～285, an anti-Ovr110 antibody.

The method for producing the antibody will be described in more detail below.
The anti-Ovr110 antibodies of the present invention are useful for treating an Ovr110-expressing cancer in a mammal or ameliorating one or more symptoms of cancer. Such cancers include ovarian, pancreatic, lung or breast cancer, urinary tract cancer, lung cancer, breast cancer, colon cancer, pancreatic cancer, and ovarian cancer, more specifically prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma Including lung adenocarcinoma, lung squamous cell carcinoma and pleural mesothelioma. Cancer includes any of the metastatic cancers described above, such as ovarian cancer metastasis, pancreatic cancer metastasis, lung cancer metastasis, and breast cancer metastasis. The antibody is capable of binding to at least a portion of cancer cells that express Ovr110 in a mammal, and preferably does not elicit or minimize a HAMA response. Preferably, the antibody is effective in vivo or in vitro when bound to Ovr110 on a cell destroys or kills Ovr110-expressing cancer cells or inhibits the growth of such tumor cells. Such antibodies include naked anti-Ovr110 antibodies (not conjugated to any agent). Naked anti-Ovr110 antibodies having in vivo tumor growth inhibitory properties include the antibodies described in the following experimental examples. Naked antibodies with cytotoxic or cytostatic properties can be further conjugated with cytotoxic agents to make them even more effective in tumor cell destruction. For example, this antibody can be conjugated to a cytotoxic agent to form an immunoconjugate as described below to confer cytotoxic properties to the anti-Ovr110 antibody. The cytotoxic agent or growth inhibitor is preferably a small molecule. Toxins such as maytansin, maytansinoid, saporin, gelonin, ricin or calicheamicin and analogs or derivatives thereof are preferred.

  The present invention provides a composition comprising an anti-Ovr110 antibody of the present invention and a carrier. For the treatment of cancer, the composition can be administered to a patient in need of such treatment, where the composition is present as one or more of the immunoconjugates or as naked antibodies. Anti-Ovr110 antibodies. Further, the composition can include these antibodies in combination with other therapeutic agents, such as cytotoxic or growth inhibitory agents including chemotherapeutic agents. The present invention also provides a formulation comprising an anti-Ovr110 antibody of the present invention and a carrier. The formulation may be a therapeutic formulation comprising a pharmaceutically acceptable carrier.

  Another aspect of the invention is an isolated nucleic acid encoding an internalizing anti-Ovr110 antibody. Included are nucleic acids encoding both heavy and light chains, and particularly hypervariable region residues, chains encoding native sequence antibodies and variants, antibody modifications and humanized forms.

  The present invention is also a method useful for treating an Ovr110-expressing cancer in a mammal or ameliorating one or more symptoms of this cancer, comprising the therapeutically effective anti-Ovr110 antibody internalizing The method is provided comprising administering an amount to the mammal. The antibody therapeutic composition can be administered for a short period (acute) or chronically or intermittently as directed by the physician. Also provided is a method of inhibiting the growth of Ovr110 expressing cells and killing it. Finally, the invention also provides kits and articles of manufacture comprising at least one antibody of the invention, preferably at least one internalizing anti-Ovr110 antibody of the invention. Kits containing anti-Ovr110 antibodies have applications in the detection of Ovr110 expression, or in therapeutic or diagnostic assays, eg, for Ovr110 cell killing assays, or for purification and / or immunoprecipitation of Ovr110 from cells. Found. For example, for isolation and purification of Ovr110, the kit can include an anti-Ovr110 antibody coupled to a solid support, such as a tissue culture plate or beads (eg, sepharose beads). Kits comprising antibodies for the detection and quantification of Ovr110 in vitro, for example in ELISA or Western blot, can be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent or radioactive label.

Production of anti-Ovr110 antibodies The following describes exemplary techniques for the production of antibodies useful in the present invention. Some of these approaches are further described in Example 1. The Ovr110 antigen to be used for the production of antibodies may be, for example, a full-length polypeptide or a portion thereof, including a synthetic peptide for a soluble form of Ovr110 lacking a transmembrane sequence or a selected portion of the protein. .

Alternatively, cells that express Ovr110 on their cell surface (eg, CHO or NIH-3T3 cells transformed to overexpress Ovr110; ovary, pancreas, lung, breast or other Ovr110 expressing tumor cell lines), Alternatively, antibodies can be generated using membranes prepared from such cells. The nucleotide and amino acid sequences of human and mouse Ovr110 are available as indicated previously. Ovr110 can be produced recombinantly in and isolated from prokaryotic cells, such as bacterial cells, or eukaryotic cells, using standard recombinant DNA methods. Ovr110 can be tagged (eg, an epitope tag) or expressed as another fusion protein to facilitate its isolation and identification in various assays.
Antibodies or binding proteins that bind to various tags and fusion sequences are available as detailed below. Other forms of Ovr110 useful for generating antibodies will be apparent to those skilled in the art.

Tags Various tag polypeptides and their respective antibodies are well known in the art. Examples include polyhistidine (poly-his) or polyhistidine glycine (poly-his-gly) tag; influenza HA tag polypeptide and this antibody 12CA5 (Field et al., Mol. Cell. Biol., 8: 2159- 2165 (1988)); c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)); and herpes simplex virus A glycoprotein D (gD) tag and this antibody (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)) are included. The FLAG peptide (Hopp et al., BioTechnology, 6: 1204-1210 (1988)) is recognized by an anti-FLAG M2 monoclonal antibody (Eastman Kodak Co., New Haven, CT). Purification of the protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix containing an anti-FLAG M2 monoclonal antibody covalently linked to agarose (Eastman Kodak Co., New Haven, CT). Other tag polypeptides include KT3 epitope peptides [Martin et al., Science, 255: 192-194 (1992)]; α-tubulin epitope peptides (Skinner et al., J. Biol. Chenz., 266: 15163-15166 (1991)); and T7 gene protein peptide tags (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990)).

Polyclonal antibodies Polyclonal antibodies are preferably raised in animals, preferably non-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to link the relevant antigen to a protein that is immunogenic in the species to be immunized (especially when using synthetic peptides). For example, an antigen is conjugated to keyhole limpet hemocyanin (KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (linked by cysteine residues), N Can be coupled using hydroxysuccinimide (depending on lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ or R ¹ N═C═NR, where R and R ¹ are different alkyl groups . Conjugates can also be made in recombinant cell culture as protein fusions.

  Animals are mixed with antigen, immunogenic conjugates or derivatives, eg 5-100 pg protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and the solution intradermally In addition, immunization can be achieved by injection at multiple sites. One month later, the animals are boosted by subcutaneous injection at multiple sites with 1/5 to 1/10 of the initial amount of peptide or conjugate in complete Freund's adjuvant. The animals are bled after 7-14 days and the serum is assayed for antibody titer. Animals are boosted until the titer is level. In addition, an aggregating agent such as alum is preferably used to enhance the immune response.

Monoclonal antibodies Monoclonal antibodies are first produced using the hybridoma method described by Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Pat. No. 4,816,567). be able to. In hybridoma methods, a mouse or other suitable host animal, such as a hamster, can be immunized as described above to produce or produce antibodies that specifically bind to the protein used for immunization. To reveal possible lymphocytes. Alternatively, lymphocytes can be immunized in vitro. Following immunization, lymphocytes are isolated and then fused with a “fusion partner” such as a myeloma cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies Principles and Practice, pp 103 (Academic Press, 1986)).

  The hybridoma cells thus prepared are seeded into and grown in a suitable culture medium, which preferably inhibits the growth or survival of unfused fusion partners such as parental myeloma cells. Or it contains two or more substances. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), selective culture media for hybridomas typically include hypoxanthine, aminopterin and thymidine. (HAT medium), this substance prevents the growth of HGPRT-deficient cells.

  Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high level production of antibodies by the selected antibody-producing cells, and are sensitive to selective media that select against unfused parent cells. There is something. Preferred myeloma cell lines are those derived from mouse myeloma lines such as MOPC-21 and MPC-II mouse tumors available from Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives such as American type. X63-Ag8-653 cells available from Culture Collection, Rockville, Maryland USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

  Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107: 220 (1980). After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be generated in vivo as ascites tumors in animals, eg, i. p. Can be grown by injection.
Monoclonal antibodies secreted by subclones can be obtained from culture media, ascites fluid or serum using conventional antibody purification procedures such as affinity chromatography (eg, using protein A or protein G-Sepharose) or ion exchange chromatography, hydroxylapatite Separation is preferably performed by chromatography, gel electrophoresis, dialysis and the like.

  The DNA encoding the monoclonal antibody is readily isolated using conventional procedures (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the murine antibody). Sequenced. Hybridoma cells act as a preferred source of such DNA. After isolation, the DNA is placed in an expression vector, which is then prokaryotic or eukaryotic host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, that do not otherwise produce antibody proteins. Alternatively, myeloma cells can be transformed or transfected to obtain monoclonal antibody synthesis in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding an antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Phickthun, Immunol. Revs., 130: 151- Includes 188 (1992).

  In addition, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Each isolation is described. Subsequent publications constitute the production of high affinity (nM range) human antibodies by chain mixing (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as a very large phage library. Combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)) have been described as a method for this. These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

  Antibody-encoding DNA can be modified to produce chimeric or fusion antibody polypeptides, such as human heavy and light chain constant region (CH and CL) sequences, with homologous mouse sequences. By substitution (US Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81: 6851 (1984)), or an immunoglobulin coding sequence is converted to a non-immunoglobulin polypeptide (heterologous Modification by fusing with all or part of the coding sequence for the polypeptide. Non-immunoglobulin polypeptide sequences can be replaced with the constant region of an antibody, or they can be replaced with the variable region of one antigen combining site of an antibody to have one antigen with specificity for one antigen. Chimeric bivalent antibodies are made that contain a combination site and other antigen combination sites with specificity for different antigens.

Humanized antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable region. Humanization is essentially according to the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), and can be performed by replacing the hypervariable region sequence with the corresponding sequence of the human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein significantly smaller than intact human variable regions are replaced by corresponding sequences from non-human species. Yes. In practice, humanized antibodies typically have some hypervariable region residues and possibly some FR residues replaced by residues from similar sites in rodent antibodies. It is a human antibody.

  Selection of both light and heavy human variable regions to be used in generating humanized antibodies reduces antigenicity and HAMA response (human anti-mouse antibody) when intended for human therapeutic use of antibodies. It is extremely important to According to the so-called “best fit” method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable region sequences. A human V region sequence closest to that of rodents is identified, of which the human framework region (FR) is accepted for humanized antibodies (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Other methods use a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).

  It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of parent sequence analysis and various conceptual humanized products using a parent and humanized sequence three-dimensional model. To do. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.

Computer programs are available that illustrate and display possible three-dimensional conformation of selected immunoglobulin candidate sequences. Examination of these designations allows analysis of the effects of potential residues on the function of the immunoglobulin candidate sequence, ie, analysis of residues that affect the ability of the immunoglobulin candidate to bind to this antigen. In this way, FR residues can be selected and combined from the recipient and the sequence transferred to achieve the desired antibody characteristic, eg, increased affinity for one or more target antigens. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
Various forms of the humanized anti-Ovr110 antibody are contemplated. For example, a humanized antibody may be an antibody fragment, such as a Fab, optionally combined with one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

Human antibodies As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (eg, mice) that, when immunized, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain junction region (J _H ) gene in chimeric germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array to such germline mutant mice results in the production of human antibodies upon antigen challenge. For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); US Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all GenPharm); see 5,545,807; and alternatively, phage display techniques (McCafferty et al., Nature 348: 552-553 ( 1990)) can be used to produce human antibodies and antibody fragments in vitro from an immunoglobulin variable (V) region gene repertoire from non-immunized donors. According to this approach, antibody V region genes are cloned in-frame into filamentous bacteriophages, eg, either major or minor major coat protein genes of Ml3 or fd, and function on the surface of phage particles. As a typical antibody fragment. Since filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that exhibit these properties. This phage thus mimics some of the properties of B cells. Phage display methods can be performed in a variety of ways and are reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V gene segments can be used for phage display methods. Clackson et al., Nature, 352: 624-628 (1991) isolated various arrays of anti-oxazolone antibodies from a small disordered combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from non-immunized human donors can be constructed, and antibodies against various arrays of antigens (including self-antigens) are essentially produced by Marks et al., J. Mol. Biol. 222. : 581-597 (1991) or Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Nos. 5,565,332 and 5,573,905. As noted above, human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

Antibody Fragments In some situations, there are advantages to using antibody fragments rather than complete antibodies. The smaller size of the fragments can allow for rapid clearance and provide improved access to solid tumors. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived from proteolytic digestion of intact antibodies (eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab′-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab) 2 fragments (Carter et al., Bio / Technology 10: 163-167). (1992)). In other methods, F (ab) 2 fragments can be isolated directly from recombinant host cell culture. Fab and F (ab) 2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described in US Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. The selected antibody may also be a single chain Fv fragment (scFv). See WO 93/16185; US Pat. No. 5,571,894 and US Pat. No. 5,587,458. Fv and sFv are the only species with an intact combination site that lacks a constant region; therefore, they are suitable for reduced non-specific binding during in vivo use. An sFv fusion protein can be constructed to obtain an effector protein fusion at either the amino or carboxy terminus of the sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, for example, as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the Ovr110 protein. Other such antibodies can combine the Ovr110 binding site with binding sites for other proteins. Alternatively, anti-Ovr110 arms can be linked to trigger molecules on leukocytes such as T cell receptor molecules (eg C133) or Fc receptors for IgG (FcγR) such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). In combination with the arm that binds to the trigger molecule above, the cellular defense mechanism can be concentrated and localized in Ovr110 expressing cells. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express Ovr110. These antibodies have an Ovr110 binding arm and an arm that binds a cytotoxic agent (eg, saporin, anti-interferon alpha, vinca alkaloid, ricin A chain, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab) 2 bispecific antibodies). WO 96/16673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies, and US Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-FcγRI antibodies. Bispecific anti-ErbB2 / Fcα antibodies are shown in WO98 / 02463. US Pat. No. 5,821,337 teaches bispecific anti-ErbB2 / anti-CD3 antibodies.

  Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one is an exact duplex. Has a specific structure. Accurate molecular purification, usually performed by affinity chromatography steps, is somewhat cumbersome and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to another method, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant region sequences. Preferably, the fusion is with an Ig heavy chain constant region comprising at least part of the hinge region, C _H 2 region, and C _H 3 region. It is preferred to have a first heavy chain constant region (CHI) containing the site necessary for light chain binding and present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, optionally, the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host cell. This allows greater flexibility in adjusting the mutual ratio of the three polypeptide fragments in an embodiment that provides an optimal yield of the desired bispecific antibody by the different ratios of the three polypeptide chains used in the structure. Sex is obtained. However, if high yields are obtained as a result of equal ratio expression of at least two polypeptide chains, or if this ratio does not significantly affect the yield of the desired chain combination, 2 Coding sequences for one or all three polypeptide chains can be inserted into a single expression vector.

  Preferably, the bispecific antibody in this method comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm ( Providing a second binding specificity). This asymmetric structure has been found to easily separate the desired bispecific compound from the undesired immunoglobulin chain combination, which means that only one half of the bispecific molecule has an immunoglobulin light chain. This is because the existence provides an easy separation method. This method is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

  According to another method described in US Pat. No. 5,731,168, an interface between a pair of antibody molecules is designed to maximize the percentage of heterodimers recovered from recombinant cell culture. Can do. A preferred interface includes at least a portion of the CH3 region. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). A compensatory “cavity” that is the same or similar in size to one or more large side chains, but has a smaller large amino acid side chain on the interface of the second antibody molecule. Created by substitution with (eg alanine or threonine). This provides a mechanism to increase the yield of the heterodimer over other undesired end products such as homodimers.

  Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin and the other can be bound to biotin. Such antibodies target immune system cells, for example, against unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089) Has been proposed to. Heteroconjugate antibodies can be made using any convenient crosslinking method. Suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980 along with a number of crosslinking techniques.

  Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure that proteolytically cleaves intact antibodies to produce F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize neighboring dithiols and prevent intermolecular disulfide formation. The resulting Fab 'fragment is then converted to a thionitrobenzoate (TNB) derivative. Next, one of the Fab′-TNB derivatives is converted back to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab′-TNB derivative to obtain a bispecific antibody. Form. The produced bispecific antibody can be used as an agent for selective immobilization of an enzyme.

  Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the production of two fully humanized bispecific antibody F (ab ') molecules. Each Fab 'fragment was secreted separately from E. coli and was subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed can bind to cells that overexpress the ErbB2 receptor and normal human T cells and can induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets. did it.

  Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked by gene fusion to the Fab 'portion of two different antibodies. Antibody homodimers are reduced at the hinge region to form monomers and then oxidized again to form antibody heterodimers. This method can also be used for the production of antibody homodimers.

The “diabody” approach described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provides an alternative mechanism for generating bispecific antibody fragments. It was. This fragment contains VH that binds to VL with a linker that is too short to allow pairing between two regions on the same chain. Thus, the VH and VL regions of one fragment are forced to pair with the complementary VL and VH regions of the other fragment, thereby forming two antigen binding sites. Other methods for making bispecific antibody fragments by using single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994).
Antibodies with a valency greater than 2 are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

Multivalent antibodies Multivalent antibodies can be internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibody of the present invention may be a multivalent antibody (eg, a tetravalent antibody) having three or more than four antigen-binding sites (other than the IgM class), which is a polypeptide chain of the antibody. Can be readily produced by recombinant expression of a nucleic acid encoding. A multivalent antibody can comprise a dimerization region and three or more antigen binding sites. Preferred dimerization regions include (or consist of) an Fc region or a hinge region. In this scenario, the antibody comprises an Fc region and three or more antigen binding site amino termini for the Fc region. Preferred multivalent antibodies herein comprise (or consist of) 3 to about 8, but preferably 4 antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable regions. . For example, one or more polypeptide chains, VDl (X 1) n -VD2- (X2) can contain n-Fc, wherein, VD1 is a first variable region, the VD2 The second variable region, Fc is one polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, one or more polypeptide chains can include: VH-CHI-flexible linker-VH-CHI-Fc region chain; or VH-CHI-VH-CHI-Fc region chain . The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein can include, for example, from about 2 to about 8 light chain variable region polypeptides. The light chain variable region polypeptides contemplated herein comprise a light chain variable region, optionally further comprising a CL region.

Other amino acid sequence modifications One or more amino acid sequence modifications of the anti-Ovr110 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-Ovr110 antibody are prepared by introducing appropriate nucleotide changes into the anti-Ovr110 antibody nucleic acid, or by peptide synthesis.
Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence of the anti-Ovr110 antibody. If the final construct has the desired characteristics, any combination of deletion, insertion and substitution is performed to arrive at the final construct. Amino acid changes can also alter the post-translational process of anti-Ovr110 antibodies, eg, changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of anti-Ovr110 antibodies that are preferred positions for mutagenicity is as described by Cunningham and Wells in Science, 244: 1081-1085 (1989). , Referred to as “alanine scanning mutagenicity”. Here, a group of residues or target residues within the anti-Ovr110 antibody (eg, charged residues such as arg, asp, his, lys and glu) are identified and neutral or negatively charged amino acids ( Most preferably, substitution with alanine or polyalanine) affects the interaction of amino acids with the Ovr110 antigen.
The amino acid positions that are functionally susceptible to substitution are then refined by introducing further or other variants at or for the site of substitution. Thus, while the site for introducing an amino acid sequence change is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenicity is performed at the target codon or region and the expressed anti-Ovr110 antibody variants are screened for the desired activity.

  Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide comprising 100 or more residues, and single or multiple amino acids Includes intrasequence insertion of residues. Examples of terminal insertions include an anti-Ovr110 antibody having an N-terminal methionyl residue, or an antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-Ovr110 antibody molecule include a fusion to the N- or C-terminus of the anti-Ovr110 antibody to an enzyme (eg, for ADEPT), or a fusion to a polypeptide that increases the serum half-life of the antibody. .

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue substituted in the anti-Ovr110 antibody molecule with a different residue. The site most relevant to substitutional mutagenicity includes the hypervariable region, but FR changes are also contemplated. Conservative substitutions are shown in Table I under the heading of “preferred substitutions”. Where such substitution results in a change in biological activity, introduce a more substantial change, named “exemplary substitution” in Table I, or further described below with respect to amino acid class. The product can then be screened for the desired properties.

Substantial modifications in the biological properties of the antibody include (a) the structure of the polypeptide backbone in the region of substitution, eg, as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c In these effects on maintaining the bulk of the side chains), this is achieved by choosing significantly different substitutions. Naturally occurring residues are grouped based on common side chain properties:
(1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser, thr; (3) Acidic: asp, glu; (4) Basic: asn, gin, his, lys, arg; (5) residues affecting chain orientation: gly, pro; and (6) aromatics: trp, tyr, phe.

  Non-conservative substitutions involve exchanging one element of these groups for another. Any cysteine residue that is not involved in maintaining the proper conformation of the anti-Ovr110 antibody can also generally be replaced with serine, improving the oxidative stability of the molecule and preventing abnormal cross-linking. Conversely, one or more cysteine bonds can be added to an antibody to improve its stability (particularly where the antibody is an antibody fragment, eg, an Fv fragment).

  A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg a humanized or human antibody). In general, the resulting variant or variants selected for further development will have improved biological properties relative to the parent antibody that produces them. A convenient way to generate such substitutional variants involves affinity maturation using phage display methods. Briefly, several hypervariable region sites (eg 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are presented in a monovalent manner as fusions from filamentous phage particles to the gene III product of Ml3 packaged within each particle. The phage-displayed mutants are then screened for their biological activity (eg, binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and human Ovr110. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. After such variants have occurred, the panel of variants is screened as described herein to select antibodies for superior development in one or more related assays. can do.

  Other types of amino acid variants of the antibody alter the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody. Antibody glycosylation is typically N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for enzymatic coupling of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide creates an effective glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine, but 5-hydroxyproline or 5- Hydroxylysine can also be used. By altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites), the addition of glycosylation sites to the antibody is conveniently achieved. The Changes can also be made by addition or substitution of one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

  Nucleic acid molecules encoding amino acid sequence variants of the anti-Ovr110 antibody are prepared by a variety of methods known in the art. These methods include isolation from natural sources (for naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis, and mutation of anti-Ovr110 antibodies. This includes, but is not limited to, preparation by cassette mutagenesis of early-prepared nucleic acid molecules that encode non-mutant forms.

  It may be desirable to modify the antibodies of the present invention with respect to effector function, for example to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. . This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, one or more cysteine residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization ability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). . Alternatively, antibodies can be designed that have dual Fc regions, which can have increased complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

  To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” means an epitope of the Fc region of an antibody.

Techniques for generating screening antibodies for antibodies with the desired properties have been described above. Furthermore, if desired, antibodies with certain biological properties can be selected.
The growth inhibitory effect of the anti-Ovr110 antibody of the present invention can be evaluated by methods known in the art, for example, using cells expressing Ovr110 endogenously or following transfection of the Ovr110 gene. it can. For example, tumor cell lines and Ovr110 transfected cells provided in Example 1 below are treated with anti-Ovr110 monoclonal antibodies of the present invention at various concentrations for several days (eg, 2-7 days) to produce crystal violet or MTT Or can be analyzed by some other colorimetric assay. Another method of measuring proliferation is by comparing ³ H-thymidine uptake by cells treated in the presence or absence of anti-Ovr110 antibodies of the invention. After antibody treatment, the cells are harvested and the amount of radioactivity introduced into the DNA is quantified in a scintillation counter. Suitable positive controls include treatment of selected cell lines with growth inhibitory antibodies known to inhibit the growth of the cell line. In vivo inhibition of tumor cell growth can be determined by various methods, as described in the Experimental Examples section below. Preferably, the tumor cell is one that overexpresses Ovr110. Preferably, the anti-Ovr110 antibody has a cell growth of Ovr110-expressing tumor cells in vitro or in vivo of about 25-100 compared to untreated tumor cells at an antibody concentration of about 0.5-30 μg / ml. %, More preferably about 30-100%, and even more preferably about 50-100% or 70-100%. Growth inhibition can be measured in cell cultures at antibody concentrations of about 0.5-30 μg / ml or about 0.5 nM-200 nM, where growth inhibition is the exposure of tumor cells to antibody 1 Determine after 10 days. The antibody may be administered by administration of anti-Ovr110 antibody at about 1 μg / kg body weight to about 100 mg / kg body weight within about 5 to 3 months, preferably within about 5 to 30 days from the initial administration of the antibody, It is growth inhibitory in vivo if it results in a decrease in tumor cell growth.

  In order to select antibodies that induce cell death, loss of membrane integrity as indicated by, for example, propidium iodide (PI), trypan blue or 7AAD uptake can be assessed relative to controls. PI uptake assays can be performed in the absence of complement and immune effector cells. Ovr110 expressing tumor cells are incubated with medium alone or medium containing, for example, about 10 μg / ml of the appropriate monoclonal antibody. Cells are incubated for a period of 3 days. Following each treatment, the cells are aliquoted into 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) that are washed and capped with a 35 mm filter to remove cell clumps. . The tube is then given PI (10 μg / ml). Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Antibodies that induce statistically significant levels of cell death as determined by PI uptake can be selected as cell death-inducing antibodies.

  For example, in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) to screen for antibodies that bind to epitopes on Ovr110 that are bound by related antibodies such as the Ovr110 antibody of the present invention. A routine cross-blocking assay can be performed as is done. This assay can be used to determine whether a test antibody binds to the same site or epitope as an anti-Ovr110 antibody of the invention. Alternatively or additionally, epitope mapping can be performed by methods known in the art. For example, antibody sequences can be mutagenized, such as by alanine scanning, to identify contact residues. Mutant antibodies are first tested for binding with polyclonal antibodies to ensure proper folding. In different methods, peptides corresponding to various regions of Ovr110 can be used in competition assays with test antibodies, or with test antibodies and antibodies with characterized or known epitopes.

  For example, a method for screening for antibodies that bind to an epitope bound by an antibody of the invention can comprise combining an Ovr110-containing sample with a test antibody and an antibody of the invention to form a mixture; The level of Ovr110 antibody bound to Ovr110 in the mixture is then determined and compared to the control mixture for the level of Ovr110 antibody bound in the mixture, where it binds to Ovr110 in the mixture compared to the control. The level of Ovr110 antibody determined is an indication of the binding of the test antibody to the epitope bound by the anti-Ovr110 antibody of the invention. The level of Ovr110 antibody bound to Ovr110 is determined by ELISA. The control may be a positive control or a negative control or both. For example, the control may be a mixture of Ovr110, an Ovr110 antibody of the invention and an antibody known to bind to the epitope bound by the Ovr110 antibody of the invention. The anti-Ovr110 antibody is labeled with a label as disclosed herein. Ovr110 may be bound to a solid support, such as a tissue culture plate or bead, such as sepharose beads.

Immunoconjugates The present invention also relates to therapy using immunoconjugates comprising an antibody conjugated to an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent.
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described previously. Conjugates of antibodies and one or more small molecule toxins such as calicheamicin, maytansinoids, trichoten and CC1065 and derivatives of these toxins having toxin activity are also contemplated herein.

Maytansine and maytansinoids Preferably, an anti-Ovr110 antibody (full length or fragment) of the invention is conjugated to one or more maytansinoid molecules.
Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the cast African shrub Maytenus serrata (US Pat. No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogs thereof are described, for example, in US Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are expressly incorporated herein by reference. To do.

Maytansinoid-antibody conjugates In an attempt to improve these therapeutic indices, maytansin and maytansinoids were conjugated to antibodies that specifically bind to tumor cell antigens. Immunoconjugates comprising maytansinoids and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures of which are incorporated herein. As clearly introduced. Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) contains a maytansinoid labeled DMI conjugated to monoclonal antibody C242 directed against human colorectal cancer. Immunoconjugates have been described. This conjugate was found to be highly cytotoxic to cultured colon cancer cells and showed antitumor activity in an in vivo tumor growth assay. Chari et al. Cancer Research 52: 127-131 (1992) includes mouse antibody A7 that binds antigen in human colon cancer cell lines, or other mouse monoclonal antibody TA. That binds to the HER-2 / neu oncogene. 1 describes an immunoconjugate in which a maytansinoid is bound via a disulfide linker. TA. The cytotoxicity of 1-maytansinoid conjugates was tested in vitro against the human breast cancer cell line SK-BR-3 expressing 3 × 10 ⁵ HER-2 surface antigen per cell. The drug conjugate achieves a similar degree of cytotoxicity as the free maytansinoid drug, which can be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-Ovr110 antibody-maytansinoid conjugate (immunoconjugate)
An anti-Ovr110 antibody-maytansinoid conjugate is prepared by chemically linking an anti-Ovr110 antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. To do. Although an average of 3-4 maytansinoid molecules bound per antibody molecule has shown efficacy in enhancing the cytotoxicity of target cells without adversely affecting antibody function or solubility, a single toxin molecule / Antibodies are expected to enhance cytotoxicity over the use of naked antibodies. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in US Pat. No. 5,208,020 and other patents and non-patent publications referred to above. Preferred maytansinoids are maytansinol and maytansinol analogs modified at other positions on the aromatic ring or the maytansinol molecule, such as various maytansinol esters.

  For making antibody-maytansinoid conjugates, including, for example, those disclosed in US Pat. No. 5,208,020 or EP Patent 0 425 235 B1 and Chari et al. Cancer Research 52: 127-131 (1992) There are many linking groups known in the art. This linking group includes disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups or esterase labile groups disclosed in the patents identified above, disulfide groups and thioethers. Groups are preferred. The conjugate of the antibody and maytansinoid can be conjugated to various bifunctional protein coupling agents such as N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate, Iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide compounds (eg Bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazonium benzoyl) ethylenediamine), diisocyanates (eg toluene 2,6 diisocyanate) and bis-active fluorinated compounds It can be made using (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 [1978]) and N to provide disulfide bonds. -Succinimidyl (2-pyridylthio) pentanoate (SPP).

  The linker may be attached at various positions to the maytansinoid molecule depending on the type of linkage. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. Preferably, the bond is formed at the C-3 position of maytansinol or maytansinol analogues.

Other immunoconjugates related to calicheamicin include anti-Ovr110 antibodies conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can cause double-stranded DNA breakage at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all American Cyanamid Company). Structural analogs of calicheamicin that can be used include γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG and θ ₁ ^I (Hinman et al. Cancer Research 53: 3336). (1993), Lode et al. Cancer Research 5 8: 2925-2928 (1998) and American Cyanamid, the aforementioned US patent specification). Another anti-tumor agent that can bind antibodies is QFA, an antifolate. Both calicheamicin and QFA have an intracellular site of action and do not easily cross the plasma membrane. Thus, the uptake of these agents into cells by antibody-mediated internalization greatly increases their cytotoxic effect.

Other cytotoxic agents Other anti-tumor agents that can bind to the anti-Ovr110 antibodies of the present invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, US Pat. Nos. 5,053,394 and 5,770,710. And the family of agents collectively known as LL-E33288 complexes as well as the esperamicins (US Pat. No. 5,877,296). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, 15 non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modelin A Chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, calcin, crotin, sapaonaria officinalis inhibitor, Gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecenes are included. See, for example, WO 93/21232 published October 28, 1993. The present invention further contemplates immunoconjugates formed between the antibody and a compound having nucleolytic activity (eg, ribonuclease or DNA endonuclease, eg, deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody can contain highly radioactive atoms. A variety of radioisotopes are useful for the production of radioconjugated anti-Ovr110 antibodies. Examples include the radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , In ¹¹¹ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , and Lu. Where the conjugate is used for diagnostics, this is a radioactive atom for scintigraphic examination, such as Tc ^99M or I ¹²³ , or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri). For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radiolabels or other labels can be introduced into the conjugate by known methods. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using a suitable amino acid precursor containing, for example, fluorine-19 instead of hydrogen. Labels such as Tc ^99M , I ¹²³ , In ¹¹¹ , Re ¹⁸⁶ , Re ¹⁸⁸ can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. Iodine can be introduced using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

  Conjugates of antibody and cytotoxic agent can be synthesized using various bifunctional protein coupling agents, for example, N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl- (N-maleimidomethyl) cyclohexane-1- Carboxylate, iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide Compounds (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6 diisocyanate) and bis-active Containing compounds (such as 1,5-difluoro-2,4-dinitrobenzene) with can be prepared. For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the binding of radionucleotides to antibodies. See WO 94/11026. The linker may be a “cleavable linker” that facilitates release of the cytotoxic agent in the cell. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers or disulfide containing linkers (Chari et al. Cancer Research 52: 127-131 (1992); US Pat. No. 5,208,020) can be used.

Alternatively, a fusion protein comprising an anti-Ovr110 antibody and a cytotoxic agent can be made, eg, by recombinant techniques or peptide synthesis. The length of the DNA includes the respective regions encoding the two parts of the conjugate that are adjacent to each other or separated by a region that encodes a linker peptide that does not destroy the desired properties of the conjugate. Can do.
In addition, an antibody can be conjugated to a “receptor” (eg, streptavidin) for use in tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient and subsequently conjugated. Unbound conjugate is removed from the circulation using a clarifying agent, and then a “ligand” (eg, avidin) that binds to a cytotoxic agent (eg, radionucleotide) is administered.

Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)
The antibody of the present invention can also be used in ADEPT by linking the antibody to a prodrug activating enzyme that converts a prodrug (eg, peptidyl chemotherapeutic agent, see WO81 / 01145) into an active anticancer drug. See, for example, WO 88/07378 and US Pat. No. 4,975,278.

  Enzyme components of immunoconjugates useful for ADEPT include any enzyme that can act on a prodrug and convert it to a more active cytotoxic form. Enzymes useful in the methods of the invention include alkaline phosphatases useful for converting phosphate-containing prodrugs to free drugs; aryl sulfatases useful for converting sulfate-containing prodrugs to free drugs; A cytosine deaminase useful for converting non-toxic fluorocytosine to the anti-cancer drug 5-fluorouracil; a protease useful for converting a peptide-containing prodrug to a free drug, such as serratia protease, thermolysin ), Subtilisin, carboxypeptidases and cathepsins (eg, cathepsins B and L); D-alanyl carboxypeptidases useful for converting prodrugs containing D amino acid substituents; free glycosylated prodrugs Charcoal useful for converting into drugs Cleaved enzymes, such as O-galactosidase and neuraminidase; P-lactamases useful for converting drugs derivatized with P-lactams to free drugs; and at these amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively. Penicillin amidases useful for converting derivatized drugs to free drugs include, but are not limited to, penicillin V amidase or penicillin G amidase. Alternatively, prodrugs of the invention can be converted into free active agents using antibodies with enzymatic activity, also known in the art as “abzymes” (eg, Massey , Nature 328: 457-458 (1987)). Enzyme-abzyme conjugates for delivering abzymes to tumor cell populations can be prepared as described herein. The enzyme of the present invention can be covalently bound to the anti-Ovr110 antibody by a technique well known in the art, for example, using the heterobifunctional crosslinking reagent described above.

  Alternatively, a fusion protein comprising at least the antigen binding region of an antibody of the invention conjugated to at least a functionally active portion of an enzyme of the invention can be obtained using recombinant DNA techniques well known in the art. (See, eg, Neuberger et al., Nature, 312: 604-608 (1984)).

Other antibody modifications Other modifications of the antibody are contemplated herein. For example, the antibody can be conjugated to one of a variety of non-protein polymers, such as one of polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or a copolymer of polyethylene glycol and polypropylene glycol. Antibodies can also be used in colloidal drug delivery systems (eg, liposomes), eg, in microcapsules prepared by coacervation techniques or by interfacial polymerization (eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmetasylate) microcapsules, respectively). , Albumin microparticles, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such an ^{approach, Remington's Pharmaceutical Sciences, 16 th} edition, Oslo, A., Ed., Disclosed in (1980).

  The anti-Ovr110 antibodies disclosed herein can also be formulated as immunoliposomes. “Liposomes” are small vesicles composed of various types of lipids, phospholipids and / or surfactants useful for delivering drugs to mammals. The components of the liposome are generally arranged in a bilayer form, similar to the lipid arrangement of biological membranes. Liposomes containing antibodies can be obtained by methods known in the art, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and W097 / 38731 published Oct. 23, 1997. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes having a desired diameter are obtained by extruding the liposomes through a filter having a predetermined pore size. Fab 'fragments of the antibodies of the invention can be conjugated to liposomes by a disulfide exchange reaction as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982). A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

Vectors, Host Cells and Recombinant Methods The present invention also provides isolated nucleic acid molecules encoding humanized anti-Ovr110 antibodies, vectors, and host cells containing nucleic acids, and recombinant techniques for producing antibodies. To do. For recombinant production of the antibody, the nucleic acid molecule encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or operable with a promoter for expression Insert into the vector in an (operable) linkage. The DNA encoding the monoclonal antibody is readily isolated using conventional procedures (eg, by using oligonucleotide probes that can specifically bind to the nucleic acid molecules encoding the heavy and light chains of the antibody). Sequenced. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. .

Signal Sequence Component The anti-Ovr110 antibody of the present invention can be produced not only directly and recombinantly, but also recombinantly as a fusion polypeptide with a heterologous polypeptide, wherein the heterologous polypeptide Is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the natural anti-Ovr110 antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, the group of alkaline phosphatase, penicillinase, lpp or a thermostable enterotoxin II leader . For yeast secretion, natural signal sequences are described in, for example, yeast invertase leader, oc factor leader (including Saccharomyces and Kluyveromycescc factor leader) or acid phosphatase leader, Calbicans glucoamylase leader, or WO 90/13646. Can be replaced by existing signals. In mammalian cell expression, mammalian signal sequences and viral secretion leaders such as herpes simplex gD signals are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the anti-Ovr110 antibody.

Origin of replication Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In general, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or self-replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. is there. In general, the origin of the replication component is not required for mammalian expression vectors (the SV40 origin can typically be used only because it contains an early promoter).

Selection Gene Component Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes confer resistance to (a) antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies, or (c) are obtained from natural media Encodes a protein that supplies critically important nutrients that are not possible, for example, this is a gene encoding D-alanine racemase for Neisseria gonorrhoeae. One example of a selection scheme uses agents that stop the growth of host cells. Those cells successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection scheme. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

  Other examples of selectable markers suitable for mammalian cells are to absorb anti-Ovr110 antibody nucleic acids such as DHFR, thymidine kinase, metallothionein-I and -11, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. It is possible to identify competitive cells for. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. A suitable host cell when using wild-type DHFR is a Chinese hamster ovary (CHO) cell line (eg, ATCC CRL-9096) that is deficient in DHFR activity.

  Alternatively, host cells transformed or co-transformed with DNA sequences encoding anti-Ovr110 antibody, wild type DHFR protein and other selectable markers such as aminoglycoside 3'-phosphotransferase (APH) (especially endogenous Wild type hosts containing DHFR) can be selected by cell growth in media containing a selection agent for a selectable marker, such as a selection agent for an aminoglycoside antibiotic such as kanamycin, neomycin or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). The trp1 gene provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4 Jones, Genetics, 85:12 (1977). Second, the presence of trp1 lesions in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids carrying the Leu2 gene.
In addition, vectors derived from the 1.6 pm circular plasmid pKDI can be used for transformation of Kluyveromyces yeast. Alternatively, an expression system for large scale production of recombinant calf chymosin has been reported for K. lactis. Van den Berg, Bio / Technology, 8: 135 (1990). A stable multicopy expression vector for the secretion of mature recombinant human serum albumin by an industrial strain of Kluyveromyces is also disclosed. Fleer et al., Bio / Technology, 9: 968-975 (1991).

Promoter Component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-Ovr110 antibody nucleic acid. Suitable promoters for use with prokaryotic hosts include the phoA promoter, P-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also include a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-Ovr110 antibody.

  Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream of the transcription start site in many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes, there is an AATAAA sequence that can be a signal to add a poly A tail coding sequence to the 3' end. All of these sequences are suitably inserted into a eukaryotic expression vector. Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase , Promoters for glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase.

  Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, are promoter regions for: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism , Metallothionein, glyceraldehyde phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

Anti-Ovr110 antibody transcription from a vector in a mammalian host cell is controlled, for example, by a promoter obtained from: if the promoter is compatible with the host cell system: from the viral genome, eg poly Omavirus, infectious epithelioma virus, adenovirus (eg adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably simian virus (simian virus) 40 ( From the genome of SV40), from heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, from heat shock promoters.
The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US Pat. No. 4,419,446. This system modification is described in US Pat. No. 4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) on expression of human P-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of Rous sarcoma virus can be used as a promoter.

Enhancer element component Transcription of the DNA encoding the anti-Ovr110 antibody of the present invention by higher eukaryotes is often enhanced by inserting an enhancer sequence into the vector. A number of enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer (bp 100-270) on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. Enhancers can be spliced in the vector at the 5 ′ or 3 ′ position to the anti-Ovr110 antibody coding sequence, but are preferably located 5 ′ from the promoter.

Transcription termination components Expression vectors used in eukaryotic host cells (nucleated cells from yeasts, fungi, insects, plants, animals, humans or other multicellular organisms) are also used to terminate transcription and mRNA Contains the sequences necessary for stabilization. Such sequences are generally available from the 5 'and optionally 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-Ovr110 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector disclosed therein.

Selection and Transformation of Host Cells As used herein, suitable host cells for cloning or expressing DNA in a vector are the prokaryotic, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria such as Gram negative or Gram positive organisms, for example Enterobacteriaceae such as Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, and Neisseria gonorrhoeae, such as B. subtilis and B. licheniformis (e.g. DD 266,710, B. licheniformis 41P disclosed in the April 12, 1989 publication), Pseudomonas Genus, for example P. aeruginosa, as well as Streptomyces are included. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W31 10 (ATCC 27,325) are preferred. These examples are illustrative rather than limiting.

  Full length antibodies, antibody fragments and antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, eg therapeutic antibodies are conjugated to cytotoxic agents (eg toxins) This is the case when the immunoconjugate alone shows efficacy in tumor cell destruction. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, a translation initiation region (TIR) and a signal sequence for optimizing expression and secretion are described, eg, US Pat. No. 5,648,237 (Carter et. Al.), See US Pat. No. 5,789,199 (Joly et al.) And US Pat. No. 5,840,523 (Simmons et al.). These patents are incorporated herein by reference. Following expression, the antibody can be isolated from the E. coli cell paste in a soluble fraction and purified by, for example, a protein A or G column, depending on the isotype. Final purification can be performed, for example, in a manner similar to the method for purifying antibodies expressed in CHO cells.

  In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Ovr110 antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234 ); Neurospora crassa; Schwanniomyces, eg Schwanniomyces occidentalis; and filamentous fungi, such as Panchobacterium, Blue mold, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger.

  Suitable host cells for the expression of glycosylated anti-Ovr110 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Corresponding permissive insect host cells from many baculovirus strains and variants and hosts such as Spodoptera frugiperda, Aedes aegypti, Aedes albopictus, Drosophila melanogaster, and Bombyx mori Has been identified. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, such viruses here being particularly mentioned as viruses of the invention It can be used for transfection of Spodoptera frugiperda cells.

  Cotton, corn, potato, soybean, petunia, tomato, Arabidopsis and tobacco plant cell cultures can also be used as hosts. Cloning and expression vectors useful in the production of proteins in plant cell cultures are known to those skilled in the art. For example, Hiatt et al., Nature (1989) 342: 76-78, Owen et al. (1992) Bio / Technology 10: 790-794, Artsaenko et al. (1995) The Plant J 8: 745-750 and Fecker et al. (1996) Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 subcloned to grow in suspension culture) Cells, Graham et al., J. Gen Virol. 36:59 (1977); pup hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells ( VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138) , ATCC CCL 75); Liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y Acad. Sci. 383: 44-68 (1982)); MRC 5 Cells; FS4 cells; and human hepatoma lineage (Hep G2).
Host cells are transformed for anti-Ovr110 antibody production using the expression or cloning vectors described above to induce promoters, select for transformants, or amplify genes encoding desired sequences In a conventional nutrient medium, modified to suit.

Host Cell Culture Host cells used to produce the anti-Ovr110 antibodies of the present invention can be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Basal Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are used to culture host cells. Suitable for. Furthermore, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or Any of the media described in US Pat. No. 5,122,469; WO 90/03430; WO 87/00195; or US Pat. No. Re. 30,985 can be used as a culture medium for host cells. All of these media may contain hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers (if desired) Eg HEPES), nucleotides (eg adenosine and thymidine), antibiotics (eg GENTAMYCIN® drugs), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent Can supplement the energy source. All other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for the host cell selected for expression and will be apparent to those of ordinary skill in the art.

Purification of anti-Ovr110 antibody When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the first step is to remove any particulate debris, host cells or lysed fragments, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. For example, protease inhibitors such as PMSF can be introduced at any of the foregoing stages to inhibit proteolysis and antibiotics can be introduced to prevent the growth of exogenous contaminants.

  Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region that is present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 region, Bakerbond ABX® resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other methods for protein purification, eg fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, anion or cation exchange Chromatography, chromatofocusing, SIDS-PAGE and ammonium sulfate precipitation on resins (eg polyaspartic acid columns) are also available depending on the antibody to be recovered.

  Following all one or more preliminary purification steps, the mixture containing the relevant antibodies and contaminants is preferably used with an elution buffer having a pH of about 2.5-4.5, preferably with a low salt concentration (e.g. Low pH hydrophobic interaction chromatography performed at about 0-0.25 M salt).

Pharmaceutical Formulations Pharmaceutical formulations of antibodies for use in accordance with the present invention are mixed with an antibody of the desired degree of purity for storage, optionally with a pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 ^th edition, Osol, A. Ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used and include the following: buffers such as acetate, tris, phosphate, citrate And other organic acids; antioxidants including ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens; For example methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins Hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; Tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes) ); And / or nonionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of 5-200 mg / ml, preferably 10-100 mg / ml.

  The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an internalizing anti-Ovr110 antibody, in one formulation, an additional antibody, such as a second anti-Ovr110 antibody that binds to a different epitope on Ovr110, or a growth factor that affects the growth of a particular cancer, for example. It may be desirable to include antibodies to other targets such as Alternatively or additionally, the composition can further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent and / or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredient can also be trapped in the prepared microcapsules, for example by coacervation techniques or by interfacial polymerization, for example colloids in hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively. Capsule delivery systems (eg liposomes, albumin microparticles, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such an approach ^{is, Remington's Pharmaceutical Sciences, 16 th} edition, Osol, are disclosed in A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and gamma ethyl- L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microparticles composed of lactic acid-glycolic acid copolymer and leuprolide acetate), As well as poly-D-(-) hydroxybutyric acid.
The formulation to be used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane.

Methods and Treatments Using Anti-Ovr110 Antibodies According to the present invention, cancers characterized by Ovr110-expressing cancer cells using anti-Ovr110 antibodies that internalize upon binding to Ovr110 on the cell surface, particularly ovarian cancer, pancreatic cancer, A subject in need thereof, having lung or breast cancer, such as ovarian serous adenocarcinoma or breast invasive ductal carcinoma, and associated metastases is treated.
Said cancer generally comprises Ovr110-expressing cells and thus anti-Ovr110 antibodies can bind to it. Although the cancers described above can be characterized by overexpression of Ovr110 molecules, the present application further provides methods for treating cancers that are not considered to be Ovr110 overexpression cancers.

  The present invention also relates to a method for detecting cells that overexpress Ovr110 and a diagnostic kit useful in detecting cells that express Ovr110 or in detecting serum of a patient. This method involves combining a cell-containing test sample with an antibody of the invention, assaying the test sample for antibodies that bind to cells in the test sample, and controlling the level of antibody binding in the test sample to control the cells. Comparing against the level of antibody bound in the sample can be included. A suitable control is, for example, a sample of normal cells of the same type as the test sample, or a cell sample known to be free of Ovr110 overexpressing cells. A level of Ovr110 binding higher than such a control sample indicates that the test sample contains cells that overexpress Ovr110. Alternatively, the control may be a sample of cells known to contain cells that overexpress Ovr110. In such cases, a level of Ovr110 antibody binding in the test sample that is similar to or greater than the control sample indicates that the test sample contains cells that overexpress Ovr110.

Ovr110 overexpression can be detected by various diagnostic assays. For example, overexpression of Ovr110 can be assayed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies can be subjected to an IHC assay to match the following criteria for Ovr110 protein staining intensity.
Score 0 staining is not observed or membrane staining is observed in less than 10% of the tumor cells.
Score 1+ a slight / barely perceptible membrane staining is detected in more than 10% of the tumor cells. Cells are only stained in some of their membranes.
Score 2+ Weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.

Score 3+ A moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
Tumors with a score of 0 or 1+ for Ovr110 expression can be characterized as not over-expressing Ovr110, while tumors with a score of 2+ or 3+ can be characterized as over-expressing Ovr110.
Alternatively or additionally, a FISH assay such as INFORM® (sold by Ventana, Arizona) or PATHVISION® (VySiS, Illinois) is performed on formalin-fixed paraffin-embedded tumor tissue Thus, the extent of Ovr110 overexpression (if any) in the tumor can be determined. Ovr110 overexpression or amplification can be assessed using in vivo diagnostic assays, eg, molecules labeled with a detectable label (eg, a radioisotope or fluorescent label) that binds Ovr110 (eg, an antibody of the invention) And the patient can be assessed by external scanning for the location of the label.

  A sample suspected of containing cells that express or overexpress Ovr110 is mixed with an antibody of the invention under conditions suitable for specific binding of the antibody to Ovr110. Binding and / or internalization of the Ovr110 antibody of the present invention indicates that the cell expresses Ovr110. The level of binding can be determined and compared to a suitable control, where a level of binding Ovr110 that is higher than the control indicates Ovr110 overexpression. A sample suspected of containing cells that overexpress Ovr110 may be a cancer cell sample, particularly a sample of ovarian cancer, eg, ovarian serous adenocarcinoma, or breast cancer, eg, breast invasive ductal carcinoma. A serum sample from the subject is also assayed for the level of Ovr110 by combining the serum sample from the subject with the Ovr110 antibody of the invention, determining the level of Ovr110 bound to the antibody, and comparing this level to the control. Where an elevated level of Ovr110 in the patient's serum relative to the control indicates overexpression of Ovr110 by the patient's cells. The subject may have cancer, such as ovarian cancer, such as ovarian serous adenocarcinoma, or breast cancer, such as breast invasive ductal carcinoma.

  Currently, depending on the stage of the cancer, treatment of ovarian cancer, pancreatic cancer, lung cancer or breast cancer includes one or a combination of the following therapies: surgery to remove cancer tissue, radiation therapy, androgen exfoliation (eg, Hormone therapy), and chemotherapy. Anti-Ovr110 antibody therapy is used in older patients who are not well tolerated with the toxicity and side effects of chemotherapy, in metastatic disease with limited radiotherapy utility, and in the management of prostate carcinoma that is resistant to androgen exfoliation treatment. Because of this, it may be particularly desirable. The tumor targeting and internalizing anti-Ovr110 antibodies of the present invention are useful for ameliorating Ovr110-expressing cancers such as ovarian cancer, pancreatic cancer, lung cancer or breast cancer during initial diagnosis of the disease or during recurrence. is there. For therapeutic applications, anti-Ovr110 antibodies can be used alone or in combination therapy, eg, in combination with hormones, anti-angiogenic agents or radiolabeled compounds, or with surgery, cryotherapy and / or radiation therapy. In combination therapy, especially for ovarian cancer, pancreatic cancer, lung cancer or breast cancer, and especially when excreted cells are not reachable. Treatment with an anti-Ovr110 antibody can also be administered sequentially in conjunction with other forms of conventional therapy, prior to or after conventional therapy, and is a chemotherapeutic agent such as Taxotere® (docetaxel) , Taxol® (paclitaxel), estramustine and mitoxantrone may be used in particularly low risk patients in the treatment of metastatic and hormone resistant ovarian cancer, pancreatic cancer, lung cancer or breast cancer it can. In this method of the invention for treating or ameliorating cancer, particularly androgen-independent and / or metastatic ovarian cancer, pancreatic cancer, lung cancer or breast cancer, one or two anti-Ovr110 antibodies are administered to cancer patients. Administration in combination with treatment with more than one prior chemotherapeutic agent. In particular, combination therapy with paclitaxel and modified derivatives (see for example EP0600517) is contemplated. An anti-Ovr110 antibody is administered with a therapeutically effective dose of a chemotherapeutic agent. Anti-Ovr110 antibodies can also be administered in combination with chemotherapy to enhance the activity and efficacy of chemotherapeutic agents such as paclitaxel. The US Pharmaceutical Handbook (PDR) discloses the dosage of the agent used in the treatment of various cancers. The dosage regimen and dosage of these aforementioned chemotherapeutic agents that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to those skilled in the art, It can be determined by a doctor.

  In particular, an immunoconjugate comprising an anti-Ovr110 antibody conjugated with a cytotoxic agent can be administered to a patient. Preferably, the immunoconjugate bound to the Ovr110 protein is internalized by the cell, resulting in an increased therapeutic effect of the immunoconjugate in killing the cancer cell to which it binds. Preferably, the cytotoxic agent targets or interferes with the nucleic acid of the cancer cell. Examples of such cytotoxic agents are as described above and include maytansin, maytansinoids, saporin, gelonin, lysine, calicheamicin, ribonuclease and DNA endonuclease.

The anti-Ovr110 antibody or immunoconjugate is administered to human patients in a known manner, for example intravenously, for example as a bolus or as a continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebral spinal cord (intracerobrospinal ), Subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or by inhalation route. The antibody or immunoconjugate can be injected directly into the body of the tumor. Intravenous or subcutaneous administration of the antibody is preferred. Other treatment regimens can be combined with the administration of anti-Ovr110 antibodies.
Combined administration includes simultaneous administration with separate formulations or a single pharmaceutical formulation, and sequential administration in either order, where both (or all) active agents are There is preferably a period of simultaneous biological activity. Preferably, such combined therapy results in a synergistic therapeutic effect.

  It may also be desirable to combine administration of one or more anti-Ovr110 antibodies with administration of antibodies directed against other tumor antigens associated with a particular cancer. Thus, the present invention also includes an antibody “cocktail” comprising one or more antibodies of the present invention and at least one other antibody that binds to other tumor antigens associated with Ovr110-expressing tumor cells. ". The cocktail can also include antibodies directed against other epitopes of Ovr110. Preferably, other antibodies do not interfere with the binding and / or internalization of the antibodies of the invention.

  The antibody therapeutic treatment methods of the present invention can include the combined administration of one or more anti-Ovr110 antibodies and one or more chemotherapeutic agents or growth inhibitory agents. Includes co-administration of cocktails. Chemotherapeutic agents include, for example, estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyurea taxanes (eg paclitaxel and doxetaxel) and / or anthracycline antibiotics Is included. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturer's instructions or as determined empirically by a skilled practitioner. Preparation and dosing regimes for such chemotherapy are also described in Chemotherapy Service, Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

The antibody is mixed with an anti-hormonal compound; such as an anti-estrogen compound such as tamoxifen; an anti-progesterone compound such as onapristone (see EP 616 812); or an anti-androgenic compound such as flutamide at a dose known for such molecules Can be matched. If the cancer to be treated is an androgen-independent cancer, the patient may have previously received anti-androgen therapy, and after the cancer becomes androgen-independent, the anti-Ovr110 antibody (and optionally herein) Other agents described in the document) can be administered to the patient.
From time to time, it may also be beneficial to co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, patients can be given surgical removal of cancer cells and / or radiation therapy before, simultaneously with, or after antibody therapy. Suitable dosages for any of the aforementioned co-administered agents are those used herein and can also be reduced due to the combined action (synergism) of the agent and anti-Ovr110 antibody.

  For prevention or treatment of disease, the dosage and mode of administration are selected by a physician according to known criteria. Appropriate doses of antibody are defined as the type of disease to be treated, severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, patient Depends on the clinical history of the patient and the response to antibodies and the physician's discretion. The antibody is suitably administered to the patient at one time or over a series of treatments. Preferably the antibody is administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, from about 1 pg / kg body weight to about 50 mg / kg body weight (eg, about 0.1-15 mg / kg / dose) of antibody, eg, one or more separate doses Or the first candidate dose for administration to a patient by continuous infusion. The dosing regimen can include administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance of a dose of about 2 mg / kg of anti-Ovr110 antibody. However, other dosage regimens may be useful. A typical daily dosage may be in the range of about 1 pg / kg to 100 mg / kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until the desired suppression of disease symptoms occurs. The progress of this therapy can be easily monitored by conventional methods and assays and based on criteria known to physicians or other skilled artisans.

  In addition to administering antibody proteins to patients, this application contemplates administration of antibodies by gene therapy. Such administration of a nucleic acid molecule encoding an antibody is encompassed by the expression “administering a therapeutically effective amount of an antibody”. See, for example, WO 96/07321 published March 14, 1996, relating to generating intracellular antibodies using gene therapy.

  There are two main methods for introducing a nucleic acid molecule (optionally contained in a vector) into a patient's cells, in vivo and ex vivo. For in vivo delivery, the nucleic acid molecule is injected directly into the patient, usually at the site where the antibody is needed. For ex vivo treatment, the patient's cells are removed, nucleic acid molecules are introduced into these isolated cells, and the modified cells are encapsulated in the patient, either directly or in a porous membrane, for example. Implanted into a patient and administered (see, eg, US Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acid molecules into viable cells. These techniques vary depending on whether the nucleic acid is transferred in vitro into cultured cells or in vivo into the intended host cells. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, and the like. A commonly used vector for ex vivo delivery of genes is a retroviral vector.

  Currently preferred in vivo nucleic acid molecule transfer techniques include viral vectors (eg, adenovirus, herpes simplex I virus or adeno-associated virus) and lipid-based systems (lipids useful for lipid-mediated transfer of genes such as DOTMA, DOPE, etc.). And DC-Chol). For a review of currently known gene marking and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992). See also WO 93/25673 and the references cited therein.

Articles of Manufacture and Kits The present invention also relates to articles of manufacture containing materials useful for the detection of Ovr110 overexpressing cells and / or the treatment of Ovr110 expressing cancers, particularly ovarian, pancreatic, lung and breast cancer. The article of manufacture comprises a container and a composition contained therein, the composition comprising an antibody of the invention. The composition can further comprise a carrier. The article of manufacture can also include a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective to detect Ovr110-expressing cells and / or treat a cancer condition and may have a sterile access port (eg, the container may be an intravenous solution bag or It may be a vial having a stopper that can be penetrated by a hypodermic needle). At least one active agent in the composition is an anti-Ovr110 antibody of the present invention. The label or package insert indicates that the composition detects Ovr110 expressing cells and / or ovarian cancer, pancreatic cancer, lung cancer and breast cancer, or more specifically ovarian serous adenocarcinoma, breast invasive ductal carcinoma, It is used to treat in patients in need. The label or package insert may further comprise instructions for administering the antibody composition to the cancer patient. Further, the article of manufacture can further include a second container containing a substance that detects the antibody of the present invention, for example, a second antibody that binds to the antibody of the present invention. This material can be labeled with a detectable label, such as those disclosed herein. The second container can contain, for example, a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. The article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

  Kits useful for various purposes are provided, such as for Ovr110 cell killing assays, to purify or immunoprecipitate Ovr110 from cells, or to detect the presence of Ovr110 in serum samples or in cell samples This kit is useful for detecting the presence of Ovr110-expressing cells. For isolation and purification of Ovr110, the kit can include an anti-Ovr110 antibody coupled to a solid support, such as a tissue culture plate or beads (eg, sepharose beads). Kits comprising antibodies for detecting and quantifying Ovr110 in vitro, eg, in an ELISA or Western blot, can be provided. For manufactured articles, the kit comprises a container and the composition contained therein, the composition comprising the antibody of the present invention. The kit can further include a label or package insert on or associated with the container. The kit can include additional components such as diluents and buffers, substances that bind to the antibodies of the invention, such as labels such as those disclosed herein, such as radioactive labels, fluorescent labels. A second antibody or enzyme that can be included can be included, or the kit can also include a control antibody. The additional component may be in a separate container in the kit. The label or package insert can provide a description of the composition and instructions for the intended in vitro or diagnostic use.

Example 1 Production and Isolation of Monoclonal Antibody-Producing Hybridoma The following MAb / hybridoma of the present invention is described below:
Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1 (previously identified as Ovr110 A22.1), Ovr110 .A77.1, Ovr110.A87.1, Ovr110.A89, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110. C5.1., Ovr110.C5.3, Ovr110.C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1 , Ovr110.C13, Ovr110.C14, Ovr110.C15, Ovr110.C16.1, Ovr110.C17.1, Ovr110.D9.1, Ovr110.I1, Ovr110.I2, Ovr110.I3, Ovr110.I4, Ovr110.I6 , Ovr110.I7, Ovr110.I8, Ovr110.I9, Ovr110.I10, Ovr110.I11, Ovr110.I13, Ovr110.I14, Ovr110.15, Ovr110.I16, Ovr110.I17, Ovr110.I18, Ovr110.I20, Ovr110 .I21, Ovr110.I22, Ovr110.J1, Ovr110.J2 and Ovr110.J3.
If the MAb has been cloned, this gets the nomenclature “X.1”. For example, the first clone of A7 is called A7.1, the second clone of A7 is called A7.2, etc. For the purposes of the present invention, reference to A7 includes all clones such as A7.1, A7.2, etc.

Immunogens and antigens (recombinant proteins, HA and His tags and transfected cells)
For the Ovr110 construct described below, nucleic acid molecules encoding regions of Ovr110 were inserted into various expression vectors to produce recombinant proteins. These nucleic acid sequences were isolated using primers whose design is routine for those skilled in the art. In some cases, the primers used are included in the following description of each component.
For illustration purposes, the predicted amino acid sequence encoded by each component is also included. However, the construct can also include a naturally occurring variant within the Ovr110 region isolated by the primer (eg, allelic variant, SNP). These variant sequences, and antibodies that bind to them, are considered to be part of the present invention, as described herein.

Ovr110A Sequence and Protein Production Full length DNA encoding the entire immature Ovr110 protein sequence of Met1-Lys282 (SEQ ID NO: 1), nucleotide sequence encoding a 17 amino acid secretion signal sequence from human stanniocalcin (STC) and a 6His tag Was inserted into a modified vector containing a sequence coding for to generate a vector encoding a recombinant Ovr110 fusion protein having a secretion signal fused to the N-terminus of the Ovr110 protein and a 6His tag fused to the C-terminus. The resulting vector was used to produce recombinant protein using standard techniques. Briefly, cells transformed with the resulting vector were cultured under conditions suitable for production of recombinant Ovr110 protein. The transformed cells are washed with Dulbecco's phosphate buffered saline (DPBS), 5 volumes containing 0.8M sodium chloride, 0.3% Zwittergent 3-14 and 0.1% octylphosphoglucoside (5 ml / cell 1 g) was dissolved in 50 mM sodium phosphate, pH 8.0 by sonication. The insoluble material was separated as a precipitate and the extraction was repeated twice. The separated precipitate was dissolved in 50 mM sodium phosphate buffer, pH 7.8, containing 6 M guanidione hydrochloride (3 ml / g cell) and the same buffer on the Akta-100 system (Amersham Biosciences, Piscataway, NJ). It was circulated through an equilibrated 10-ml-Ni-NTA (Qiagen, Alameda, CA) column at a flow rate of 5 ml / min for about 40 column volumes (CV). The column was then washed in the same phosphate-guanidine buffer with 2 CV of the same phosphate-guanidine buffer, 2 CV of 20 mM imidazole, 2 CV of 50 mM imidazole, and 4 CV of 100 mM imidazole.

  The resulting vector was used to produce recombinant protein by standard techniques. Briefly, cells transformed with the resulting vector were cultured under conditions suitable for production of recombinant Ovr110 protein. The transformed cells are washed with Dulbecco's phosphate buffered saline (DPBS), 5 volumes containing 0.8M sodium chloride, 0.3% Zwittergent 3-14 and 0.1% octylphosphoglucoside (5 ml / cell 1 g) was dissolved in 50 mM sodium phosphate, pH 8.0 by sonication. The insoluble material was separated as a precipitate and the extraction was repeated twice. The separated precipitate was dissolved in 50 mM sodium phosphate buffer, pH 7.8, containing 6 M guanidione hydrochloride (3 ml / g cell) and the same buffer on the Akta-100 system (Amersham Biosciences, Piscataway, NJ). It was circulated through an equilibrated 10-ml-Ni-NTA (Qiagen, Alameda, CA) column at a flow rate of 5 ml / min for about 40 column volumes (CV). The column was then washed in the same phosphate-guanidine buffer with 2 CV of the same phosphate-guanidine buffer, 2 CV of 20 mM imidazole, 2 CV of 50 mM imidazole, and 4 CV of 100 mM imidazole.

  Ovr110 was eluted with 4 CV 500 mM imidazole in phosphate-guanidine buffer and the column was further washed with 4 CV 50 mM sodium phosphate, pH 7.6 containing 1 M imidazole and 6 M guanidine hydrochloride. Samples from the collected fragments were subjected to SDS-PAGE and Western blot analysis to assess the purity of Ovr110A. Purified fragments were pooled and dialyzed against PBS. The precipitate was collected and resuspended in a small amount of PBS by short sonication.

Ovr110B sequence and protein production For the immunization of mice, a recombinant protein fragment of Ovr110 that constitutes only the expected extracellular part of the molecule was generated and a monoclonal antibody (MAb) that binds to the external surface of the cell was selected. . A DNA fragment encoding the Ovr110 sequence from the immature protein Gly30 (underlined in the sequence below) to Lys282 (add Met at the start codon position), including the signal peptide, was inserted into the modified vector, where The modified vector comprises a nucleotide sequence encoding a 17 amino acid secretion signal sequence from human stanniocalcin (STC) and a nucleotide sequence encoding a 6His tag, from human stanniocalcin (STC) fused to the N-terminus of Ovr110 protein. A vector encoding a recombinant Ovr110 fusion protein having a 17 amino acid secretion signal and a 6His tag fused to the C-terminus (Ovr110B).

The resulting vector was used to transform DH10Bac bacteria and generate an infectious vector by transposition. Recombinant baculovirus was then generated by transfecting Sf9 cells with the transposed vector. Recombinant Ovr110B was expressed by infecting the Hi5 cell line with amplified and harvested virus particles.
Culture medium from recombinant Hi5 cells was harvested 48 hours after infection. The medium was concentrated 10 times and diafiltered with 30 volumes of PBS pH 7.9. The diafiltered material was then incubated with 10 ml Ni-NTA rapid gel (Qiagen) at 4 ° C. overnight in the presence of a protease inhibitor cocktail. The gel was poured onto an SK column and washed with 2 CV 50 mM sodium phosphate, pH 7.8 containing 0.5 M sodium chloride. Ovr110B was eluted with graded increasing imidazole (20 mM 4 CV, 50 mM 4 CV, 100 mM 4 CV, 500 mM 4 CV and 1000 mM 2 CV) in the same phosphate-sodium chloride buffer. Samples from the collected fragments were subjected to SDS-PAGE and Western blot analysis to assess the purity of Ovr110B. The purified fragments were pooled and concentrated. The final product was dialyzed in PBS.

ＰＣＲ断片は、ＮｓｉＩおよびＮｈｅＩで消化し、ヒト・スタニオカルシン１（ＳＴＣ−１）分泌シグナルをコードするヌクレオチド配列および１０ヒスチジンタグをコードするヌクレオチド配列を含む、修飾哺乳類発現ｐＣＭＶ５Ｈｉｓ２ベクター中に、インフレームでクローニングして、ＮＨ２末端に融合したヒト・スタニオカルシン１（ＳＴＣ−１）分泌シグナルおよびＣＯＯＨ末端に融合した１０ヒスチジンタグをそれぞれ有する組換えＯｖｒ１１０タンパク質をコードする、組換えプラスミドｐＣＭＶ５ｊｏｓ２＿Ｏｖｒ１１０を産生した。Ｏｖｒ１１０配列は、以下の記載において下線で示す。ＤＮＡ配列分析は、PE Applied Biosystems （Foster City, CA）からのABI Prism BigDye終了サイクルシーケンシング簡易反応キットを用いて行った。

Sequence and Protein Production of Mammalian Cell Expressed Ovr110 Generating a PCR fragment of a nucleic acid molecule encoding Ovr110 from Gly30 to Lys282 from a shuttle vector containing full-length Ovr110 cDNA (pDONR201_Ovr110) using the following oligonucleotide primers: Generated by:

The PCR fragment was digested with NsiI and NheI and in frame into a modified mammalian expression pCMV5His2 vector containing a nucleotide sequence encoding a human stanniocalcin 1 (STC-1) secretion signal and a nucleotide sequence encoding a 10 histidine tag. Cloning produced the recombinant plasmid pCMV5jos2_Ovr110 encoding the human stanniocalcin 1 (STC-1) secretion signal fused to the NH2 terminus and the recombinant Ovr110 protein each having a 10 histidine tag fused to the COOH terminus. The Ovr110 sequence is underlined in the following description. DNA sequence analysis was performed using the ABI Prism BigDye end cycle sequencing simple reaction kit from PE Applied Biosystems (Foster City, Calif.).

The recombinant plasmid pCMV5His2_Ovr110 was used to transfect 293T cells in suspension culture (1 L serum-free medium) in spinner flasks.
The culture medium was harvested 48 hours after transfection. The medium was concentrated 10 times and diafiltered with 100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0. Concentrated medium containing Ovr110 was passed over a 5 mL nickel metal chelate column (Ni-NTA Rapid, Qiagen Inc.) pre-equilibrated with 100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0. . The column was then washed with 6 column volumes (CV) of 100 mM sodium phosphate, 400 mM NaCl, 2 mM imidazole, 10% glycerol, pH 8.0. Ovr110 was eluted from the column with 22 CV of 100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0 containing 5 mM imidazole and 500 mM imidazole, respectively. Fragments containing Ovr110 were pooled and dialyzed against 100 mM sodium phosphate, 400 mM NaCl, 5% glycerol, pH 7.5.

単離した核酸分子はベクターに挿入した；コードされたタンパク質を次に示す。

BTLA sequence and protein production:
Nucleic acid molecules encoding Met1 to Ser289 full-length human BTLA (hBTLA) were cloned by PCR from the pituitary and lymph node cDNA libraries using the following oligonucleotide primers:

The isolated nucleic acid molecule was inserted into the vector; the encoded protein is shown below.

The truncated hBTLA gene encoding Met1-Pro152 encompassing the surface immunoglobulin (Ig) region was cloned by PCR from the Burkitt lymphoma cDNA library using the following oligonucleotide primers:
ATN551: (see sequence above) SEQ ID NO: 6 and
ATN554: 5'-CG GCT AGC GGG TCT GCT TGC CAC TTC GTC (SEQ ID NO: 8).
Nucleic acid molecules encoding the full-length secreted form of MBT1-Ser241 hBTLA lacking the membrane permeation region were cloned by PCR using oligonucleotide primers ATN551 and ATN552 from a lymph node cDNA library. The PCR fragment is digested with PmeI and NheI and ligated to either pCMV5HIS2 or pCMV5Fc1 cleaved with the same enzymes to obtain a protein construct having a C-terminal extension AS-HHHHHHHHHH or AS-mouse Fc region (mFc), respectively. Generated. DNA sequence analysis was performed using the ABI Prism BigDye end cycle sequencing simple reaction kit from PE Applied Biosystems (Foster City, Calif.).

  Recombinant plasmid pCMV5Fc1_BTLA5NT encoding only the surface Ig region of hBTLA fused to mFc (BTLA5NT_mFc) was used to transfect 293T cells in suspension culture (1 L serum-free medium) in a spinner flask. The culture medium was harvested 48 hours after transfection. Sodium chloride was added to the harvested medium at 3M final and the medium was adjusted to pH 8.0. The BTLA-containing medium was then passed over a 5 mL recombinant protein A column previously equilibrated with 10 column volumes (CV) of 50 mM borate, 4 M NaCl, pH 8.0. The Protein A column was then washed with 30 CV 50 mM borate, 4 M NaCl, pH 8.0. BTLA5NT_mFc was eluted from the Protein A column with 10 CV of 100 mM acetate, pH 3.0. The BTLA5NT-mFc-containing fragment was neutralized with 1M Tris-HCl, pH 9.0 and dialyzed in 3 L PBS, pH 7.5.

Ovr107 sequence and protein production Recombinant Ovr107 protein was used to screen and select multireactive hybridoma clones. Ovr107 is widely upregulated in multiple cancers, and as used herein, recombinant Ovr107 contains a potentially cross-reactive hexahistidine tag. Therefore, recombinant Ovr107 is useful for identifying polyreactive antibodies.
A full-length cDNA encoding the Ovr107 sequence from Met1 to Ile596 (WO01 / 37864 human Ovr107 ovarian cancer marker) was cloned by PCR and inserted into a vector. The Ovr107 coding region was then transferred by recombination into a vector containing a nucleotide sequence encoding a 6His tag to generate an Ovr107 fusion protein with a 6His tag fused to the C-terminus.

得られたベクターを用いて、ＤＨ１０Ｂａｃ細菌を形質転換し、転位により感染ベクターを生成させた。次に、組換えバキュロウィルスを、Ｓｆ９細胞を転位ベクターで形質移入することにより生成した。組換えＯｖｒ１０７を、増幅し収穫した組換えバキュロウィルス粒子でＳｆ９またはＨｉ５細胞系を感染させることにより、発現させた。

The resulting vector was used to transform DH10Bac bacteria and an infectious vector was generated by transposition. Next, recombinant baculovirus was generated by transfecting Sf9 cells with a translocation vector. Recombinant Ovr107 was expressed by infecting Sf9 or Hi5 cell lines with amplified and harvested recombinant baculovirus particles.

  Recombinant baculovirus infected Hi5 cells were harvested 48 hours after infection. Cells were washed with DPBS and lysed by sonication in 100 mM sodium phosphate pH 8.0 containing 0.4 M sodium chloride, 10% glycerol, 1% Triton X-100 and 10 mM imidazole (5 ml / g cell). ). The extract was incubated with 10 mg DNase for 30 minutes at room temperature and then centrifuged at 17,000 rpm for 30 minutes in an SS-34 rotor. The supernatant was further filtered through a 45 nm filter and onto a 5 ml-Ni-NTA column (Quiagen) equilibrated with 0.1 M sodium phosphate, pH 8.1 containing 0.4 M sodium chloride and 10% glycerol. Loading was performed at a flow rate of 3 ml / min. The column was washed with 15 column volumes (CV) of the same equilibration buffer and Ovr107 was increased stepwise in phosphate-sodium chloride buffer (20 mM 10 CV, 50 mM 10 CV, 100 mM 10 CV, 500 mM was eluted at 5 CV and 1000 mM at 5 CV). Fragments were collected in 5 ml / tube and samples from the collected fragments were subjected to SDS-PAGE and Western analysis to assess the purity of Ovr107. The purified fragments were pooled and concentrated. The final product was dialyzed in PBS.

Generation of a stable LMTK mouse cell line Mammalian vectors encoding C-terminal HA-tagged Ovr110 were transfected into mouse LMTK cells. Stable transfectants were selected for 7-10 days in Dulbecco's Modified Eagle Medium (DMEM) / 10% FBS using 10 ug / ml blastocidin followed by fluorescence-based single cell sorting (Coulter Elite, Beckmann Coulter, Sunnyvale, CA) was performed at 1 cell / well in 96 well plates. Transfected LMTK cells are cells grown in 96-well plates (VWR, Brisbane, CA), expanded into 24-well plates, and then expanded into 6-well plates. After 1 week of culture, individual clones were assayed for expression of Ovr110 by Western blot using an anti-HA antibody (Covance, Richmond, Calif.). Two LMTK cell clones expressing the highest level of Ovr110-HA were expanded into 75 cm ² flasks (VWR) for hybridoma screening and cryopreserved in fetal bovine serum (FBS) containing 10% DMSO. And stored in liquid nitrogen at -196 ° C to ensure the maintenance of viable clonal cultures. The protein encoded by the vector is shown below with the HA-tag underlined.

Generation of Transient 293F Transfected Cells A nucleic acid molecule encoding Ovr110 (SEQ ID NO: 13) without an HA tag was cloned into a mammalian expression vector, PCDNA3.1, and human 293F cells ( Invitrogen) was transfected. 50 ml of 293F cells cultured in free style medium (GIBCO) at 10 ⁶ cells / ml were transfected with 293fectin transfection reagent (Invitrogen) according to the manufacturer's guidelines. DNA, cells and 293fectin were mixed in OPTI-MEM medium (GIBCO). Cells were used for analysis 48 hours after transfection.

Mice were immunized with soluble E. coli expressed Ovr110B recombinant protein for immunization A series MAb fusion. Mice were immunized with mammalian expression extracellular region of Ovr110 for C series MAb fusion. For I series MAb fusion, mice were immunized twice weekly with His-tagged Ovr110 protein from the insects described above.
Groups of 8 BALB / c mice were immunized intradermally on both hind footpads. All injections were 25 uL per paw. The first injection of 10 ug of antigen per mouse (Day 1) is Dulbecco's phosphate buffered saline (DPBS) mixed with Titermax gold adjuvant (Sigma, Saint Louis, MS) at an equal volume to volume ratio. Went in. Subsequent injections of 10 ug of antigen per mouse were performed on days 5, 9, 12, 16, 19, 23, 26, 29, 30 which consisted of 20 uL DPBS and 5 uL Adju-phos adjuvant (Accurate per mouse). Chemical & Scientific Corp., Westbury, NY). The last booster injection on day 33 consisted of antigen diluted only with DPBS. Fusion occurred on day 37.

Hybridoma fusion mice were sacrificed upon completion of the immunization protocol and draining lymph node (popliteal) tissue was collected by sterile dissection. Lymph node cells are dispersed by pressing into a DMEM through a sterile sieve and removing T cells through anti-CD90 (Thy 1.2) coated magnetic beads (Miltenyl Biotech, Baraisch-Gladbach, Germany). It was.
These lymph node cells rich in primary B cells are then electron-celled with the continuous myeloma cell line P3x63Ag8.653 (Kearney, JF et al., J. Immunology 123: 1548-1550, 1979). Immortalized by fusion (BTX, San Diego, CA). Successfully fused cells were selected by culturing in standard media containing hypoxanthine, azaserine (HA) (Sigma) (DMEM / 10% FBS). These fusion cultures were immediately dispersed in the wells of a 96-well culture plate at 10 million cells per plate. Dispersion of cultures in 96-well culture plates immediately after fusion facilitated selection of a wider range of hybridoma clones producing a single specific antibody. Supernatants from wells were screened by ELISA for reactivity against Ovr110B, Ovr110A and for the absence of cross-reactivity with an irrelevant protein (Ovr117).

  Monoclonal cultures consisting of genetically uniform progeny from single cells were subjected to flow cytometry (Coulter Elite) after the screening procedure described above, with single viable cells in the wells of two 96-well plates. Was established by classification using The resulting mouse B cell hybridoma culture was expanded using standard tissue culture techniques. Selected hybridomas were stored frozen in fetal bovine serum (FBS) with 10% DMSO and stored in liquid nitrogen at −196 ° C. to ensure maintenance of viable clonal cultures.

Screening and selection of antibody producing hybridomas Hybridoma cell lines were selected for production of Ovr110 specific antibodies by enzyme linked solid phase immunoassay (ELISA). Ovr110B or Ovr107 protein was non-specifically adsorbed to the wells of a 96-well polystyrene EIA plate (VWR). 50 uL of Ovr110B protein or peptide BSA conjugate at 0.91 mg / mL in (DPBS) was incubated overnight at 4 ° C. in the wells of a 96 well polystyrene EIA plate. Plates were washed twice with Tris buffered saline (TBST) containing 0.05% Tween 20, pH 7.4. The plate wells were then drained and non-specific binding ability was blocked by completely filling the wells with TBST / 0.5% bovine serum albumin (TBST / BSA) and incubating at room temperature (RT) for 30 minutes. . The plate wells were then drained and 50 uL of hybridoma culture medium sample was added to the wells and incubated for 1 hour at RT. The wells were then washed 3 times with (TBST). Next, 100 uL of alkaline phosphatase-conjugated goat anti-mouse IgG (Fc) (Pierce Chemical Co., Rockford, IL) diluted 1: 5000 in TBST / BSA was added to each well and incubated for 1 hour at RT. The wells were then washed 3 times with TBST. Next, 100 uL of alkaline phosphatase substrate paranitrophenyl phosphate (pNPP) (Sigma), 1 mg / mL in 1 M diethanolamine buffer, pH 8.9 (Sigma) was added to each well and incubated for 20 minutes at RT. . Bound alkaline phosphatase activity was indicated by a visible yellow color. The enzymatic reaction was quantified by measuring the absorbance of the solution at a wavelength of 405 nm. The culture that produced the highest absorbance value was selected for expansion and further evaluation.

Ovr110 MAb ELISA screening After 2 weeks of culture, hybridomas with supernatants that produced ELISA absorbance values greater than 1.0 for Ovr110B and less than 0.2 for Ovr107 were transferred from 25 96-well culture plates to new 96-well culture plates. The cells were rearranged in the culture plate and further cultured for 1 week.
After an additional week of culture, 12 hybridomas from the A series and C series with supernatants that yield ELISA absorbance values greater than 1.0 for Ovr110B (Tables 1A and 1B) and less than 0.2 for Ovr107 15 were selected by cell sorting for single cell cloning in 96-well culture plates (Coulter Elite).

Results from ELISA screening of cloned Ovr110 MAb After 2 weeks of culture, supernatants from two hybridoma clones from each parental hybridoma were greater than 1.5 for Ovr110B (Tables 1A and 1B) or Ovr110 peptide, or Ovr107. Was tested for production of ELISA absorbance values less than 0.2. The following clones: Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A72.1, Ovr110.A77.1, Ovr110.A87. 1, Ovr110.A89.1, Ovr110.A 99.1, Ovr110.A102.1, Ovr110.A107.1, Ovr110.C1, Ovr110.C2, Ovr110.C3.2, Ovr110.C4, Ovr110.C5.3, Ovr110 .C6.3, Ovr110.C7.1, Ovr110.C8, Ovr110.C9.1, Ovr110.C10.1, Ovr110.C11.1, Ovr110.C12.1, Ovr110.C13, Ovr110.C14, Ovr110.C15 , Ovr110.C16.1 & Ovr110.C17.1 were all selected for scale-up for immunohistochemistry, immunofluorescence and functional studies.

FACS screening for cell surface binding of Ovr110 MAb LMTK-Ovr110-HA stable transfectants and Ovr110 mRNA positive (SKBR3) and mRNA negative (HT29) tumor cell lines were grown in DMEM / 10% FBS + P / S. One day prior to staining, LMTK-Ovr110-HA stably transfected cells were stimulated by adding sodium butyrate to a final concentration of 5 mM. For FACS analysis, LMTK-Ovr110-HA cells or tumor cell lines were washed once with 10 ml Ca ^{+ 2} / Mg ⁺² free DPBS and then 7 ml warm (37 ° C.) Cellstripper (Mediatech, Herndon, VA ) Was added per 150 cm ² flask. The cells were then incubated at 37 ° C. for 5 minutes while tapping the flask to remove tightly attached cells. Cells were removed and pipetted several times to break up aggregates and then immediately placed in DMEM / 10% FBS / 5 mM sodium butyrate. Cells were then centrifuged for 5 minutes at 1300 rpm and resuspended in DMEM / 10% FBS / 5 mM sodium butyrate. Cells were incubated at 37 ° C. for 30 minutes recovery time. Prior to staining, cell viability was measured using a Guava Viacount (Guava Cytometers, City, Calif.) And, if> 90% viable, they were placed in a 96 well v bottom plate (VWR). Disperse and stain with MAb.

Cells were aliquoted in 96-well v-bottom plates at 0.5-1.0 × 10 ⁶ cells / well and centrifuged for 2 minutes at 1500 rpm. The supernatant is aspirated and the plate is briefly shaken on a vortex mixer to resuspend the cells and then 200 ul DPBS / 3% FBS / 0.01% Na azide (FACS buffer) is added to each Added to wells. Centrifugation and aspiration were repeated, and then a 25 uL serial dilution of the hybridoma supernatant or purified MAb was added to the cells. Plates were stored on ice for 15 minutes and then washed in 200 uL FACS buffer as described above. This washing procedure was repeated twice and then 25 uL of phycoerythrin (PE) -conjugated donkey anti-mouse IgG Fc antibody (Jackson Immunoresearch Laboratories Inc., West Grove, Pa.) Was added to the cells. After 15 minutes on ice, cells were washed twice as described above, then resuspended in 250 uL FACS buffer and analyzed on a cell sorter or flow cytometer. In some cases, 133 ul FACS buffer and 67 uL of 1% paraformaldehyde / DPBS are added to each well for fixation and then the volume is added with DPBS for storage overnight at 4 ° C. prior to analysis. Increased to 250 uL. Stained cells were analyzed on an Elite fluorescence activated cell sorter (FACS) (Beckman-Coulter, Miami, FL).

The results of a representative experiment showing cell surface expression by FACS analysis are shown in FIG. Binding of Ovr110MAb A7.1 followed by binding of donkey anti-mouse Ig-PE conjugate (DAMPE) makes 49% of Ovr110-transfected mouse LMTK cells positive and the fluorescence intensity (average fluorescence intensity, MFI) is only DAMPE. 7.5 times higher than cells stained with. Data for further FACS analysis of human tumor cell lines is shown in Table 2 below. As can be seen in this result, each of Ovr110.C3.2, Ovr110.C5.3 and Ovr110.C6.3 binds to more than 80% of fresh Ovr110 mRNA positive SKBR3 cells, whereas the control negative MAbPro4. D9.1 bound to less than 2% of cells from these same breast cancers. Ovr110.C3.2, Ovr110.C5.3 and Ovr110.C6.3 similarly bound to less than 2% of Ovr110 mRNA negative cells of colon cancer line HT29.

Supernatants from Ovr110 I-series antibody fusions were screened by ELISA assay (described above) against Ovr110 protein from insects and Ovr110-human IgFc fusion protein from 293F mammalian cells and negative control protein. 22 well cells were reactive to both insect and mammalian proteins and non-reactive to negative control proteins. When tested by flow cytometry on Ovr110 transfected 2923F cells and the non-transfected parental cell line, the five antibodies (I2, I3, I4, I11 and I20) showed high levels of reactivity with the Ovr110 cell surface protein, The parental control strain showed very little reactivity.

The isotype of Ovr110 MAb isotype MAb was determined using a commercially available immunoassay test kit for determining mouse monoclonal antibody isotype (IsoStrip, Roche Diagnostic Corp., Indianapolis, IN). The results of isotype determination are shown in Table 3. All MAbs were IgG ₁ / κ isotype except for Ovr110 MabA10.1, which is an IgG _2b / κ isotype.

Ovr110 MAb affinity analysis binding kinetics and affinity constants were calculated from surface plasmon resonance measurements using a BIACORE 3000 instrument (Biacore, Piscataway, NJ). Experiments were designed to generate simultaneously on-rate, off-rate and affinity values for Ovr110 MAb.

  Rabbit anti-mouse IgG Fc antibody (Biacore) was immobilized by standard amine coupling (Biacore) on flow cells 2, 3 and 4 of a CM5 sensor chip (Biacore). Flowing cell 1 was used as a blank surface for subtraction criteria, which was activated and then inactivated with ethanolamine. Ovr110 MAb was captured on a rabbit anti-mouse IgG Fc coated chip and antigen bound. Therefore, these measurements should show the actual 1: 1 affinity, rather than the avidity effect observed with direct immobilization of the antigen, due to the divalent nature of IgG antibodies. The MAb was diluted to 15 ug / mL with HBS EP buffer (Biacore) and divided into multiple tubes to minimize evaporation between cycles. MAb passed through the flowed cells at 20 uL / min for 2 minutes. MAb capture levels ranged from 200 to 300 response units (RU) per flow cell. After capture of the MAb, the surface was allowed to stabilize for 3 minutes. The Ovr110B (1.56 mg / mL) antigen is then flowed over the captured MAb at 20 uL / min in flowing cells and in blank flowing cells for 4 min in order, with the following concentrations: 144, 72, 36, Passed at 18, 9, 4.5 ug / mL. Since the Ovr110B molecular weight is 35 kD, these antigen concentrations correspond to 4.11, 2.06, 1.03, 0.514, 0.257, 0.129 uM. Two repeated cycles were performed for each antigen concentration or buffer. There is a 420 second dissociation time between cycles, and chip or blank surface regeneration for anti-mouse IgG Fc antibody is performed by running 100 mM glycine at pH 1.75 through the flowed cells for 30 seconds at 100 ul / min. It was.

The obtained data was analyzed by BiaEvaluation software (Biacpre) using global fit simultaneous ka / kd assuming Langmuir binding. The software Rmax parameter was set locally to compensate for minor changes in the anti-mouse IgG Fc capture phase. The calculated affinity is shown in Table 4, which is in the range of 10 ⁻⁹ to 10 ⁻¹³ and is high enough to achieve a therapeutic dose of 10 mg / kg or less in vivo.

Western Blot Protein extracts for Western blot analysis were prepared in cell lysis buffer (1% NP-40; 10 mM sodium phosphate, pH 7.2; 150 mM sodium chloride) in Ovr110-293T transfectants and mammals. Prepared from an adenocarcinoma cell line. Proteins were separated in a Novex-XCell II Minicell gel apparatus (Invitrogen, Life Tech) under denaturing conditions by electrophoresis on NuPAGE 4-12% Bis-Tris gel (Invitrogen Life Technologies, Carlsbad, Calif.) And then PVDF The membrane was transferred using an XCell II Blot Module (Invitrogen Life Technologies). Following protein transfer, the membrane was blocked in 1% blocking reagent (Roche Diagnostic Corp., Indianapolis, IN) and purified primary antibodies (Ovr110 monoclonal antibodies: A10.2, A13.1, A31.1, A57.1, A72.1, A77.1, A89, A107, C3.2, C5.1, C5.3, C6.3, C7.1, C9.1, C11.1, C12.1 or C17. 1) and then with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.) overnight at 4 ° C. and finally by chemiluminescence by ECL Advanced Western Blotting Detection Kit (Amersham Biosiences, Visualized using Piscataway, NJ).

  Deglycosylation experiments were performed on protein extracts from Ovr110-293T transfectants, Ovr110 mRNA positive (QPCR +) and Ovr110 mRNA negative (QPCR−) mammalian adenocarcinoma cell lines and ovarian cancers (New England). Biolabs, Inc., Beverly, MA) according to the manufacturer's instructions. The deglycosylated sample was then analyzed by Western blot as described above. Briefly, 100 ug protein extract was denatured at 100 ° C. for 10 minutes in glycoprotein denaturation buffer (0.5% SDS + reducing agent). This was followed by the addition of kit reaction buffer at a final concentration of 1% NP-40 and 50 mM sodium phosphate and incubation at 37 ° C. for 4 hours before adding 100 units of PNGaseF.

The results of the Western blot experiment are summarized in Table 5A and Table 5B. As can be seen, Ovr110MAb A10.1, A13.1, A31.1, A57.1, A72.1, A77.1, A89, A107, C3.2, C5.1, C5.3, C6.3 , C7.1, C9.1, C11.1, C12.1 and C17.1, the expected size minor for non-glycosylated Ovr110 protein (30 kDa) in lysates of Ovr110-transfected human 293T cells And a major band of 49-60 kDa was identified. The large band was consistent with the presence of several glucosylation sites on the Ovr110 protein. A major band of 50-60 kDa was also detected in lysates from the QPCR + human breast cancer cell lines SKBR3 and MCF7 (ATCC, Manassas, Va.) With the same Ovr110 MAb, but the QPCR cell lines CaOV3 and HT29 (ATCC ) Was not detected in the lysate. Deglycosylation with PNGase resulted in a band detected by Ovr110 MAbA57.1 in lysates from Ovr110 transfected human 293T cells and in lysates from SKBR3 and MCF7 (ATCC) breast cancer cell lines ˜60 kDa (glycosylated ) To ˜30 kDa (presumed aglycosylated size). Deglycosylation of lysates from three ovarian cancer samples with PNGase F also reduced the size of the band detected by Ovr110 MAb 57.1 from ˜60 kDa (glycosylated) to ˜30 kDa.

Example 2: Cell surface binding of Ovr110 MAb in live cancer cells as shown by immunofluorescence The following cancer cell lines were used in this study: ovarian OvCar-3, ovarian CaOV-3 and breast SKBr-3. OvCAR-3 and SKBR-3 cells express Ovr110, whereas control CaOV-3 cells do not express Ovr110.
Cells were seeded on 18 mm glass coverslips and cultured for 48 hours at 37 ° C. in DMEM containing 10% fetal calf serum and penicillin and streptomycin, and then treated with anti-Ovr110 MAb.

  Eleven Ovr110 MAbs (Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A77.1, Ovr110.A87.1, Ovr110.A89 .1, Ovr110.A99.1, Ovr110.A102.1 and Ovr110.A107.1) were tested to determine which antibodies bind to the cell surface of Ovr110-expressing cancer cells. Primary MAb was added to the medium at a final concentration of 10 ug / ml and incubated at 37 ° C. for 1 hour. After fixation with 3% formaldehyde in phosphate buffered saline (PBS), cells were incubated with secondary Cy3-labeled donkey anti-mouse (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 minutes at a concentration of 10 ug / ml. did. After washing, the cells were placed in a medium containing DAPI (Vectastain, Vector, Burlingame, CA), visualized the cell nuclei to provide counterstaining, and observed with a Zeiss fluorescence microscope Axiophot equipped with an appropriate fluorescence filter . Micrographs were obtained with a CCD camera.

Results The 11 MAbs tested (Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A77.1, Ovr110.A87.1, Of Ovr110.A89.1, Ovr110.A99.1, Ovr110.A102.1 and Ovr110.A107.1), 10 antibodies were able to bind to at least a part of Ovr110-expressing cells. FIG. 2 shows the binding of Ovr110.A57.1 to the cell membrane of OvCAR-3 ovarian cancer cells (arrows in FIG. A) and SKBR-3 breast cancer cells (arrows in FIG. B). The cell membrane of CaOV-3 cells, a control cell line that does not express Ovr110, was not labeled when the cells were incubated with the same antibody (FIG. 2C).

Binding and internalization in living cancer cells This test was performed using fluorescent antibodies. By labeling the antibody with the fluorescent dye Cy3, antibody binding and internalization can be visualized by fluorescence microscopy. This approach is well established in the art. OvCAR-3 cells that do not express Ovr110 were used as negative controls.

Cy3 binding
Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1, and Ovr110.A87.1 were labeled with Cy-3. Cy3 conjugation was performed according to standard procedures and manufacturer guidelines. Briefly, 1 mg of antibody is dialyzed against 0.1 M bicarbonate buffer (pH 9.3) for 60 minutes, mixed with Cy3 dye, incubated for 2 hours at RT, and then Pierce Slide- Transferred into A Lyzer dialysis cassette and dialyzed in 2 liters of PBS for 6 hours at 4 ° C. This operation was repeated 6 times. Cy3-conjugated antibodies were collected and the concentration was measured with a spectrometer at 280 nm.
Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1 and Ovr110.A87.1 MAb were then added together with the cells at a concentration of 10 ug / ml at 37 ° C. in a water chamber. Incubated for 60 minutes, washed in PBS and fixed with 3% formaldehyde in PBS for 10 minutes. Following fixation, cells and coverslips were placed in medium containing DAPI (Vectastain) to visualize cell nuclei and cells were observed using a Zeiss fluorescence microscope Axiophot equipped with an appropriate fluorescence filter. Micrographs were obtained with a color CCD camera.

Results Cancer cells expressing Ovr110 by immunofluorescence microscopy of cancer cells treated with Cy3-Ovr110.A7.1, Ovr110.A13.1, Ovr110.A72.1, Ovr110.A57.1 and Ovr110.A87.1 Have been shown to bind to and internalize fluorescent antibodies to different extents. FIG. 3A (arrow) shows that Cy3-Ovr110.A57.1 is internalized by SKOV-3 cells and to a lesser extent by SKBr-3 cells (FIG. 3B) after binding. In CaOV-3 control cells, no Cy3-Ovr110.A57.1 binding was observed or a low degree of binding was observed (FIG. 3C). A feature of internalization pattern staining in SKOV-3 cells is the presence of perinuclear vesicles, likely corresponding to endosomes located close to the Golgi apparatus (FIGS. 3A and 3B).

Conclusion Ovr110 MAb is internalized upon binding to Ovr110 on the cell surface of Ovr110-expressing cancer cells in vitro.
Ovr110 distribution in tumors and normal tissues assessed by immunohistochemistry Tissues Formalin-fixed paraffin-embedded blocks of breast cancer, ovarian cancer and normal adjacent tissues were obtained from the National Disease Research Interchange (Philadelphia, PA). Normal organ OCT-embedded blocks were obtained from Zoion (Hawthorne, NY).

Immunohistochemical staining of formalin-fixed paraffin-embedded sections 6 μm thick sections cut from formalin-fixed paraffin-embedded blocks are heated at 45 ° C., deparaffinized in Histoclear and re-watered with a series of ethanol to PBS It was summed up. For antigen retrieval, section slides are boiled in 10 mM sodium citrate buffer (pH 6.0) at 120 ° C. and 15-17 PSI for 10 minutes in a decloaking chamber (Biocare, Walnut Creek, Calif.). It carried out by. Endogenous peroxidase activity was stopped by treating sections with 3% hydrogen peroxide solution for 15 minutes. Slides were incubated with 1% BSA to block non-specific antibody binding and then DAKO autostainer (1 hour at room temperature using 6 different primary Ovr110 MAbs at a concentration of 1 ug / ml). Dako Co., Carpinteria, (CA). After washing in Tris buffered saline (TBS) containing 0.5% Tween-20, the slides were incubated with anti-mouse IgG conjugated to horseradish peroxidase (HRP) as a secondary antibody. After washing in TBS containing 0.5% Tween-20, sections were visualized by treatment with 3,3′-diaminobenzidine chromogen (Immunovision Technologies, Co. Daly City, Calif.) For 2-5 minutes. Then, it was counterstained with hematoxylin, and after dehydration, it was placed in Permount medium. Normal mouse IgG at the same concentration as the primary antibody served as a negative control.

Immunohistochemical staining for OCT-embedded frozen unfixed sections Slides were cut to 5-8 um at the appropriate temperature in a cryochamber and allowed to air dry at room temperature for at least 30 minutes. IHC was performed using the Immunovision Powervision Kit (Immunovision Technologies, Co. Daly City, CA). Briefly, slides were washed in TBS to remove OCT and incubated with primary antibodies Ovr110.A13.1 and Ovr110.A57.1 for 1 hour at room temperature. These were then post-fixed for 10 minutes in 4% paraformaldehyde fixative and processed as described above.

result
Ovr110.A7.1, Ovr110.A10.1, Ovr110.A13.1, Ovr110.A57.1 and Ovr110.A87.1 were used to immunolabel ovarian serous adenocarcinoma clinical sample sections. FIG. 4 shows the distribution of Ovr110 in ovarian tumors evaluated by IHC using Ovr110.A57.1.
Of the 15 clinical samples, 13 (87%) showed positive immunolabeling with Ovr110.A57.1, while 14 of 15 (93%) used Ovr110.A13.1. (Table 6A). Specific immunostaining was limited to epithelial cells in tumors, and the number of positive cells varied from 50% to almost all tumor cells. 4A and 4B, it can be seen that tumor epithelial cells show strong membrane staining (arrows) and weaker cytoplasmic staining, with no background staining in the stroma. FIG. 4C shows that there is no specific label in a control experiment in which the primary antibody is replaced with a mouse IgG fragment.

  When Ovr110.A57.1 is used, 8 out of 10 breast cancer clinical samples (80%) are positive, and when Ovr110.A13.1 is used, 5 out of 10 (50%) are positive. It was positive (Table 6A). FIG. 5 shows the pattern of expression in clinical samples of breast invasive ductal carcinoma. Labeling was limited to the surface of tumor epithelial cells (FIGS. 5A and 5B, arrows). FIG. 5C shows that there is no specific label in a control experiment in which the primary antibody is replaced with a mouse IgG fragment. The expression level for Ovr110 in neoplastic ovary and breast tissue was high as judged by the intensity of the immunolabel. A limited number of pancreatic cancer samples were tested for Ovr110 expression. By Ovr110.A57.1, 2 out of 4 clinical samples (50%) showed Ovr110 expression, and Ovr110.A13.1 showed 3 out of 5 Ovr110 expression (Table 6A). Figures 6A and 6B show the immunolabeling pattern obtained with Ovr110.A57.1 in a clinical sample of pancreatic adenocarcinoma. Labeling is limited to the cell surface of epithelial cells (arrows). No specific labeling was observed when normal mouse IgG was used instead of Ovr110.A57.1. Lung cancer tissue was also found to be positive for immunolabeling with A7.1, A13.1 and A31.1 (in 2/2, 2/3 and 1/2 cases, respectively).

Ovr110 expression was also analyzed in normal tissues and was generally found to be negative in the following organs: liver, stomach, bladder, testis, colon, ovary, prostate and lung (only by A13.1 1 / 7 was positive). Normal heart cells showed moderate cytoplasmic staining, but no cell surface staining. The kidney showed moderate membrane staining in several terminal tubules and the ascending loop. The apical membrane in normal breast and pancreatic ducts was also labeled.

  Ovr110 binding to rodent homologues in normal mouse mammary tissue prepared, sectioned and stained in the same manner as normal human tissue to facilitate preclinical safety testing for binding of anti-Ovr110 MAb Several anti-Ovr110 MAbs were tested. The results of this test are shown in Table 6B. Ovr110.A57.1, Ovr110.C3.2, Ovr110.C6.3 and Ovr110.C12.1 all responded to the ductal epithelium of the mouse mammary gland in a pattern similar to that in normal human breast tissue.

Summary The results show that Ovr110 expression can be used as a sensitive and specific indicator for ovarian serous adenocarcinoma and breast invasive ductal carcinoma, provided that Ovr110 is used in some pancreatic and lung cancers. And some anti-Ovr110 MAbs apparently also reacted with related molecules in mouse mammary tissue. The cell membrane staining pattern indicates that Ovr110 is an ideal therapeutic target.

Example 3: Killing of Ovr110-transfected CHO cells by incubation with MAb and anti-mouse MAb saporin conjugate Experiments were performed as follows: CHO cells transfected with Ovr110 (Ovr110-CHO) were transformed into Mab-zap. Incubation with Ovr110 Mab premixed with goat anti-mouse Ig saporin conjugate (Advanced Targeting Systems, San Diego, Calif.) And cell survival was measured after 72 and 96 hours to determine the potential killing effect on these Ovr110 expressing cells. Detected. On day 1, Ovr110-CHO cells were placed in 96-well flat bottom sterile cell culture plates (Corning) in triplicate wells at 2000 cells / 75 uL / well in F12 medium containing 10% FBS, P / S. did. Plates were incubated overnight at 37 ° C., 5% CO2. Duplicate plates were set up for reading after 72 and 96 hours. Day 2 (0 hour), 25 uL 4x final MAb concentration only, or 25 uL 4x MAb pre-mixed with 25 uL 4x MabZap, or 25 uL 4x MabZap alone, or 25 uL medium only Triplicate was added to the wells of a 96-well plate to make a final volume of 100 uL. The final concentration of MAb was 2 ug / mL, 0.4 ug / mL, 0.08 ug / mL and 0 ug / mL, and the final concentration of MAbZap was 1 ug / mL. Medium only, MAb only (2 ug / mL only), and MAbZap only triplicate wells were used as negative controls. The anti-transferrin receptor MAb5E9 (ATCC, Manassus, VA) was used as a positive control MAb for killing. The plate was gently shaken for 5 minutes to mix the reagents and then incubated at 37 ° C., 5% CO 2. Day 5 (72 hours), 10 uL of Alamar Blue stock solution (Biosource International, Camarillo, Calif.) Is added to the wells of the first plate set, which is added at 37 ° C., 5% CO 2 for 2-7. Incubated for hours. Plates are then analyzed on a SpectraMAX GeminiEM spectrophotometer (Molecular Devices, Sunnyvale, Calif.) (Emission = 590 nm, excitation = 560 nm and auto cutoff = 570 nm) and viability is expressed as a percentage of medium-only control wells. It was.

Ovr110.A10.1, Ovr110.A31.1, Ovr110.A57.1, Ovr110.A87.1, Ovr110.C3.2, Ovr110.C5.1, Ovr110.C5.3, Ovr110.C6.3, Ovr110. The test results of C9.1, Ovr110.C11.1 and Ovr110.C12.1 are shown in Table 7 . As can be seen here, MAbZap alone resulted in high background values and inhibited Ovr110-CHO cell proliferation by 0-41.4%. This did not occur in negative control wells with only Pro104-CHO cells and MAbZap, in these cases 0-10% growth inhibition (data not shown). However, none of the Ovr110 MAb alone produced growth inhibition over 3.8% of Ovr110-CHO cells. On the other hand, when MAbZap saporin conjugate was added, all Ovr110 MAbs tested produced more than 10% growth inhibition compared to MAbZap alone. In particular, Ovr110.A57.1 resulted in 15.4-21.1% higher inhibition of Ovr110-CHO cell proliferation than MAbZap alone at concentrations of 0.08, 0.4 and 2.0 ug / mL with MAbZap Resulted in 57.7-63.4% higher growth inhibition compared to only wells. In conclusion, growth inhibition of Ovr110-expressing CHO cells was obtained at MAb concentrations that were readily achievable in vivo for therapeutic purposes. These in vitro data suggest that the Ovr110 MAb is suitable for targeting drugs or isotopes to tumor cells in vivo.

Example 4: Binding of Ovr110 MAb and soluble BTLA-Fc to activated T cells and tumor cells Anti-human B7x / B7H4 and anti-mouse B7S1 MAbs were previously shown to bind to activated T cells (Prasad et al., Immunity 18: 863-73 (2003); Sica et al., Immunity 18: 849-61 (2003); Zang et al., Proc. Nat.1 Acad. Sc.i US A. 100: 10388 -92 (2003)). To confirm binding of the inventive Ovr110 MAb to activated cells, fresh human T cells were purified and stimulated with different compounds as described below. The binding of Ovr110 MAb to CD3 positive activated T cells expressing CD25 (IL-2R) and CD71 (TFR) was analyzed by FACS. The claim that BTLA is a putative receptor for Ovr110 (B7x / B7H4) (Watanabe et al., Nat Immunol. 2003 4: 670-9; Carreno & Collins Trends Immunol. 2003 24: 524-7) and this book The binding of the human BTLA-mouse IgG2aFc fusion disclosed in the specification to these activated T cells and tumor cells was examined.

Preparation of human peripheral blood leukocytes (PBL) Human peripheral blood from normal male donors was obtained from a spontaneous donor at the Stanford Blood Center (Palo Alto, CA). Mononuclear cells were separated using standard Ficoll / Hypaque single step density gradient centrifugation (1.077 g / mL).

Activation of T cells Mononuclear cells at a final concentration of 10 ⁶ / mL for 3 days at 37 ° C., 10% CO ₂ , in one of the following to PRMI-1640 (CellGro) in a standard 25 cm ² tissue culture flask Plus 10% FCS (Hyclone, Utah), 10 ug / mL phytohemagglutinin (PHA-M) (Sigma, St. Louis, MO) or 10 ug / mL lipopolysaccharide (LPS) (Sigma), or Combination of 10 ng / mL phorbol myristic acetate (PMA) (Sigma) and 1 uM ionomycin (Sigma).

Immunofluorescence and flow cytometry cells were collected after 3 days of PHA stimulation and washed well. Mononuclear cells were dispersed in 96 well V-bottom plates and incubated in autologous serum to block Fc receptors. An anti-CD3 FITC antibody (Serotec, Raleigh, NC) is added to each well and either CD80PE, CD86PE, CD25PE, or biotinylated anti-CD71 (Serotec), Ovr110.A57.1 or Ovr110.C3.2 is added to the second. MAb was added at 20 ug / mL for two-color analysis. Cells were washed twice and phycoerythrin-streptavidin (PESA) was added to wells preincubated with biotinylated MAb. Cells were washed twice and resuspended in FACS buffer. Cells were preincubated in autologous serum and stained with Ovr110-Ig or BTLA-Ig fusion protein 20 ug / mL. The cells are washed twice and donkey anti-mouse PE (1 ug / mL) is added to the sample, then the cells are washed twice and incubated in mouse serum to block free binding sites on the donkey anti-mouse antibody. did. Anti-CD3 FITC antibody was then added as a final step to identify T cells. After two washes, the cells were resuspended in FACS buffer and analyzed by flow cytometry. Human tumor cell line SKBR3 was incubated with MAb or BTLAFc as described above.

All samples were analyzed with an EPCS Elite flow cytometer. All histograms were generated using the Winmdi program. CD3-positive T cells were used as gates to analyze the expression of the B7 family and activation markers (CD71 and CD25).

  Increased expression of CD25 and CD71 compared to unstimulated T cells, as seen in FIG. 7 where the black curve shows binding of MAbs to unstimulated T cells and Tables 8A, 8B, 8C ( That is, an increase in PE average fluorescence value) was achieved on PHA activated T cells (gated on CD3). Increased expression of CD71 (ie, positive cells or increased fluorescence intensity) was achieved with activated dendritic cells (gated on CD1c) and activated mononuclear cells. These data indicate positive activation of T cells, dendritic cells and mononuclear cells. The fluorescence profiles of FIG. 7 and Tables 8A, 8B, and 8C show that CD86 (B7.2) and Ovr110 (MAbA57.1) expression is increased in activated T cells and activated dendritic cells, and Ovr110 is activated Shows some increase in nuclear cells.

  To assess whether BTLA is a receptor for OVr110, the binding of BTLA-Fc (mouse IgG2a) fusion protein to Ovr110 transfected 293F cells (Ovr110-293F) was tested. From FIGS. 7G, 7H and 7I, some BTLA-Fc fusion protein bound to Ovr110-293F cells (17% positive, MFI24.57) but not to control 93F cells (2% Cells are positive, MFI3.44) can be observed. Furthermore, no detectable binding of mouse IgG2a to Ovr110-293F cells via the Fc fragment could be observed (3% of the cells were positive, MFI 4.32). From the data of BTLA-Fc and Ovr110-Fc binding to activated cells shown in Table 8B, and the binding of BTLA-Fc to Ovr110-293F cells in FIGS. 7G, 7H and 7I, Ovr110 expression and BTLA It can be concluded that there may be an association between binding to Ovr110 expressing cells. BTLA may be a receptor for Ovr110, or expression of Ovr110 may promote BTLA binding through complex formation, signal transduction or other cellular processes. In any case, these two proteins can be useful as diagnostic or therapeutic agents by blocking tumor function. In addition, modified forms of BTLA-Fc and Ovr110-Fc bound to, for example, cytotoxic or cytostatic components, or other functionalities can also be used as therapeutic agents.

  The data shown in Tables 8A and 8B indicate that MAb A57.1 selectively binds to activated T cells and that MAb C3.2 selectively binds to the tumor cell line SKBR3. These data suggest that there are different epitopes on Ovr110 on T cells and Ovr110 on tumor cells. This minimizes any immunosuppressive effect caused by the binding of Ovr110 on T cells while binding Ovr110 to tumor cells and reduces the immunosuppressive effect of tumor-expressed Ovr110 or shed Ovr110 This is advantageous. Antibodies such as Ovr110.C3.2, or antibodies that bind to epitopes bound by C3.2 are useful as therapeutic anti-tumor antibodies.

Example 5: Confirming the function of Ovr110
Materials and methods Cells and cell cultures RK3E, 293T, IEC-18, SKOV3, HeLa, CaOV3, HT29, MCF7 and SKBR3 cell lines were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in DMEM (Invitrogen) with L-glutamine and 4.5 g / L glucose supplemented with 10% FBS and 100 U / mL penicillin / streptomycin (Cellgro). All cells were kept at 5% CO ₂ in a humidified 37 ° C. incubator.

Western Blot FIG. 8A is a Western blot detection of Ovr110 protein using mAb A57.1 in a cell line and shows the correlation between mRNA expression and protein expression. The Western blot in FIG. 8B shows detection of Ovr110 protein in cell lines and human ovarian tumor tissue samples, but no detection in normal adjacent tissues (NAT). Western blot of FIG. 8 C, which shows the detection of Ovr110 protein in cell lines and human breast tumor tissue samples, no detectable in normal adjacent tissues (NAT). In addition, FIG. 9 is a Western blot showing that Ovr110 protein is not detected in extracts of major organs, and that therapeutic strategies directed to Ovr110 will not interfere with major or critical organ functions To suggest.

siRNA Oligonucleotide Design and Preparation To design siRNA molecules, sequences were selected from the open reading frame of Ovr110 mRNA based on previously described methods (Elbashir et al., 2001). Disordered “scrambled” siRNA sequences that did not cause any known cellular mRNA knockdown were used as negative controls. As an additional negative control, siRNA targeting Emerin was used to demonstrate that knockdown of non-essential mRNA had no effect on Ovr110 levels or any biological endpoint tested (data shown). Not) As a positive control for mRNA knockdown resulting in apoptosis induction, siRNA targeting DAXX was used based on published data (Michaelson et al., J. Cell Sci., 2003 Jan 15; 116 (Pt2 ): 345-52). A BLAST search against the human genome was performed for each selected siRNA sequence to ensure that the siRNA was target specific and had no function of knocking down other sequences. All siRNA molecules (HPP purified class) were chemically synthesized by Xeragon Inc. (Germantown, MD). Prior to use, siRNA was dissolved in sterile buffer, heated at 90 ° C. for 1 minute, and then incubated at 37 ° C. for 1 hour. The siRNA oligonucleotide with two thymidine residues (dTdT) at the 3 ′ end of the sequence consisted of the following specific RNA sequence:

Transfected with siRNA oligonucleotides 6 × 10 ⁴ SKBR3 cells were seeded in 12-well plates 18-24 hours prior to transfection. Transient transfections were performed using Oligofectamine reagent (Invitrogen) according to the manufacturer's protocol. A final concentration of 100 nM siRNA (excluding DAXX siRNA which was 200 nM) and 1.5 ul oligofectamine were used per well of cells. For all experiments, siRNA was transfected in triplicate. Evaluation of parallel wells of cells was performed 72 hours after transfection by quantitative real-time RT-PCR (QPCR) for changes in mRNA levels, by Western immunoblot for changes in protein levels, and for changes in apoptosis. Two different assay systems (see below) were performed. FIG. 10A shows that Ovr110 siRNA is specific for the Ovr110 transcript and does not knock down GAPDH as measured by QPCR. FIG. 10B shows that Ovr110 siRNA reduces Ovr110 message expression in the absence and compared to the scrambled siRNA control. The results showing the down-regulation of Ovr110 protein are shown in FIG. SiRNA # 37 against Ovr110 was also tested with cells that do not express Ovr110 and no effect on apoptosis was seen (data not shown). All findings were confirmed by at least two additional experiments.

Quantitative real-time RT-PCR (QPCR)
The QuantiTech SYBR Green RT-PCR kit from Qiagen Inc. was used for QPCR evaluation. 20-40 ng of template RNA was used per reaction. QPCR was performed using the Taqman 7700 sequence detection system (Applied Biosystem Inc.).

Apoptosis assay Two different assay kits were used to assess the effect of siRNA on apoptosis. Using the "Apo-ONE Homogeneous Caspase-3 / 7 Assay" kit (Promega Inc.), the test cells are directly solubilized in the culture plate and the caspase activity reflected in the fluorescence reading is determined according to the supplier's instructions. It was measured. Using a second kit, “Guava Nexin V-PE Kit” (Guava Technologies Inc.), the treated cells were harvested by trypsinization and washing, and about 10 ⁵ cells were transferred to 40 ul of the provided buffer. Resuspend in solution and add 5 ul of Annexin V (+) and 7-AAD (-) each. Following a 20 minute incubation on ice, cells were analyzed using a Guava PCA flow cytometer according to the manufacturer's instructions. 12 and 13 show the results showing that apoptosis is induced by Ovr110 knockdown.

  For the anoikis assay, IEC-18 and RK3E cells expressing the specified gene were trypsinized and resuspended in FBS-free medium at a density of 150,000 and 200,000 cells / ml, respectively. A 1 ml aliquot of the mixture was plated into each well of a 12-well plate and the sample was incubated at 37 ° C. for 24 hours. Cells were then harvested and evaluated using the Guava Nexin V-PE Kit described above. Ras, a strong oncogene, was used as an anoikis assay positive control and AP was used as a negative control. The results are shown in FIG.

SDS-PAGE and Western immunoblot analysis 72 hours after transfection with siRNA, cell extracts were placed on ice with lysis buffer (1% NP40, 10 mM Na ₂ PO ₄ , 0.15 M NaCl) and protease inhibitor cocktail. (Roche Inc.). Other experimental extracts using virus-infected cells or cells without transfection were prepared in a similar manner. Proteins extracted from harvested tumors were snap frozen and ground in extraction buffer (50 mM Tris-HCl, pH = 7.2, 150 mM NaCl, 0.5% IG-Pal plus protease inhibitor). Tumor cells were prepared by homogenization and subsequently sonicated and then centrifuged in a microcentrifuge tube to clean the extract. Between 20-50 ug of protein extract was used for each gel lane: protein equivalent concentrations were evaluated to compare protein levels on the same gel. The clarified extract is mixed with an equal volume of 2 × concentrated Laemmli sample buffer (Invitrogen), heated to 70 ° C. for 10 minutes, and then precast 4-12% SDS-polyacrylamide minigel (Nupage, Invitrogen). Analyzed using MES running buffer (Nupage, Invitrogen). The gel was transferred to an Immobilon-P PVDF membrane (0.45 μm pore size, Invitrogen) using 1 × Nupage transfer buffer and 10% methanol. The membrane was washed and blocked with 5% nonfat dry milk in PBS containing 0.05% Tween-20 for 1 hour at room temperature. Membranes were incubated with primary antibody overnight in 5% nonfat dry milk in PBS containing 0.05% Tween-20. A mouse monoclonal antibody directed against Ovr110 was produced using recombinant Ovr110 protein. Monoclonal antibody against Ovr110 was used at a final concentration of 1 ug / ml and mouse monoclonal antibody against GAPDH (Chemicon Inc.) was used at a final concentration of 2 ug / ml. Following primary antibody incubation, the membranes were washed 4 times in 1 × PBS each containing 0.05% Tween-20 for 10 minutes at room temperature. Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Jackson Lab Inc.) was used in 5% non-fat dry milk in PBS containing 0.05% Tween-20 for 1 hour at room temperature (1: 10,000 dilution). ) Primary monoclonal antibody was detected. The membrane is finally washed four times for 10 minutes in 1 × PBS with 0.05% Tween-20, followed by detection using enhanced chemiluminescence (ECL) reagent according to manufacturer's instructions (Amersham) did.

Expression Vector Construction For expression of Ovr110 protein in mammalian cells, Ovr110 cDNA was subcloned into the pLXSN vector (BD Bioscience / Clontech) and the sequence was confirmed. The pLXSN retroviral vector utilizes the MLV LTR to drive the cloning of cDNA expression into a number of cloning sites, and the SV40 promoter drives the expression of the Neo gene encoding G418 resistance. PLAPSN, a retroviral expression vector encoding alkaline phosphatase (AP), was purchased from BD Bioscience / Clontech (pLXSN-AP).

Virus production Narrow host viruses were used to infect RK3E and IEC-18 cells, and broad host viruses were used to infect SKOV3 cells. For narrow host virus packaging, 293T cells were seeded on Biocoat collagen-coated plates (BD) at a density of 8 × 10 ⁵ cells per well of a 6-well dish one day prior to transfection. Cells were transfected with purified plasmid DNA using Lipofectamine plus PLUS reagent (Invitrogen). 0.8 μg viral plasmid DNA per cell well: pLXSN-Ovr110, pLXSN-Ovr110HA or pLXSN-AP, 0.8 μg pVpack-Eco and 0.8 μg pVpack GP (Stratagene), 125 μL DMEM without serum And 10 μL of PLUS reagent stock, followed by incubation for 15 minutes at room temperature. Subsequently, 8 μL Lipofectamine diluted in 125 μL DMEM medium was added to the DNA / PLUS reagent mixture and incubated for 15 minutes at room temperature. 1 ml of DMEM was added to the final lipofectamine / DNA mixture and applied to a cell monolayer already containing 1 ml of DMEM but no serum, followed by incubation at 37 ° C. for 3 hours. The transfection mixture was replaced with DMEM containing 20% FBS and the cells were grown overnight. Finally, the medium was replaced with DMEM supplemented with 10% FBS and 100 U / mL Pen / Strep to collect the virus. Virus containing media was harvested after 24 hours and filtered through a 0.45 μm polysulfone filter. The same procedure was repeated for broad host virus packaging, except that the pVpack Ampho plasmid (Stratagene) was used instead of the pVpack Eco plasmid.

Virus infection and selection Polybrene (Hexadimethrine Bromide; Sigma) was added to fresh virus-containing medium at a final concentration of 4 μg / ml. RK3E, IEC-18 or SKOV3 cells plated at a density of 3 × 10 ⁵ cells per 100 mm ² dish the previous day were washed once with phosphate buffered saline (Cellgro) containing Ca 2+ and Mg 2+. Viral solution (6 ml per 100 mm ² dish) was applied directly to the cells and then incubated for 3 hours in a humidified 37 ° C. incubator with 5% CO ₂ with occasional swirling. The virus-containing medium is replaced with fresh growth medium and the cells are incubated at 37 ° C. for 60-72 hours, at which point a final concentration of 350 ug / mL G418 sulfate (Cellgro) is included in the growth medium. Cells infected with the virus were selected. The cells were maintained at 70-80% confluence and the G418-containing medium was changed every 2 days. Following G418 selection, the pool of cells was used for subsequent experiments including validation of Ovr110 protein expression by Western immunoblot analysis, where cells were extracted and analyzed as described above. AP expression by the infected cell monolayer was monitored by staining, whereby the cell monolayer was fixed with 0.5% glutaraldehyde solution at room temperature for 10 minutes, washed with PBS, and heated at 65 ° C. for 30 minutes. APs were visualized by incubating with BCIP / NBT liquid substrate (Sigma) for 2-3 hours.

Nude mice were injected subcutaneously with a retrovirally infected G418 selection pool of SKOV3 cells expressing either tumor xenograft experiments AP or Ovr110. Parental SKOV3 cells were also used for comparison. 10 ⁷ cells of each type were transplanted to each of 6 mice with Matrigel. Mice injected with tumor cells developed 100% tumors, and tumor formation was monitored every 4 days during the study period by palpation and, if possible, caliper measurements. The results are shown in FIG. Data are expressed as mean group tumor volume over time.

Example 6: Ovr110 Monoclonal Sandwich ELISA Detection High binding polystyrene plates (Corning Life Sciences (MA)) were coated overnight at 4 ° C. with 0.8 μg / well anti-Ovr110 MAb. The coating solution was removed by aspiration and free binding sites were blocked by adding 300 μl / well Superblock-TBS (Pierce Biotechnology, Illinois) and 100% calf serum for 1 hour at room temperature (RT). After washing 4 times with TBS + 0.1% Tween 20, 50 μl of assay buffer (TBS, 1% BSA, 1% mouse serum, 1% calf serum, 0.1% Tween 20) is added to each well, then 50 μl of antigen was added and incubated for 90 minutes. For checkerboard experiments, each pair was tested on 50 ng / ml and 0 ng / ml recombinant mammalian Ovr110 (extracellular). For each sandwich ELISA, standards of 10, 2.5, 0.5, 0.25, 0.1 and 0 ng / ml Ovr110 were run in parallel with the test sample. Standards and test samples were diluted with assay buffer. For detection, 100 μl of biotinylated MAb (1 μg / ml) was added to each well and incubated for 1 hour at room temperature with shaking. After washing, 100 μl horseradish peroxidase-conjugated streptavidin (1 mg / ml, Jackson ImmunoReserach Laboratories, PA) was added to each well at a 1: 20,000 dilution and incubated at RT for 30 minutes with shaking. After washing, the plates were then expressed for 30 minutes at RT using DAKO TMB Plus substrate (DAKO, Denmark). The reaction was stopped with 100 μl / well of 1N HCl and the plate was read at 405 nm using a Spectramax 190 plate reader (Molecular Devices, CA).

  For the checkerboard ELISA, all possible combinations of antibodies were tested for efficiency as a coating or detection reagent. The following pairs: A72.1 / A7.1, A77.1 / A57.1, A57.1 / A7.1 and A57.1 / C3.2 give the best signal / noise ratio and are further improved by sandwich ELISA Evaluated to analyze the efficiency of detection of endogenous Ovr110 in lysates from cancer cell lines and body fluids. The 2700 serum samples listed below were tested using the A72.1 / A7.1 pair.

Results Table 9A and 9B show the results of a checkerboard ELISA that tested 10 MAbs of the A series and 8 MAbs of the C series. Each antibody was tested as both a coating antibody and a detection antibody in all possible combinations. All pairs were tested in duplicate on 100 ng of recombinant Ovr110B protein in buffer, with buffer alone as a blank. Results are shown as specific signal / noise ratio. Based on these pairing data, MAb detected two different epitopes. Ovr110 A7, A77, A87 and A10 MAbs react with one or more epitopes close enough to sterically hinder the binding of the other three MAbs. All C series antibodies also detect this epitope (or overlapping epitopes). One or more other different epitopes are detected by Ovr110 A89, A57, A31, A72, A107 MAb. Several pairs with the greatest signal / noise ratio were used to test for sensitivity to recombinant protein and reactivity to native protein in cell lines and some initial serum samples.

これらの表の結果から得られるＯｖｒ１１０ＭＡｂのエピトープマップを、図１６に示す。

The epitope map of Ovr110 MAb obtained from the results of these tables is shown in FIG.

Human serum samples Human cancer samples and benign serum samples were obtained from IMPATH-BCP, Inc and DSS (Diagnostic Support Service). Serum samples from healthy women were obtained from ProMedex, LCC. All samples were aliquoted upon arrival and stored at −80 ° C. until use.

Results As noted above, a sensitive detection system based on the use of horseradish peroxidase (HRP) and sensitive TMB substrate (DAKO) was used for detection of Ovr110 in serum samples. The minimum detectable dose (MDD) for Ovr110 in this ELISA format is 100 pg / ml. For the median calculation, a sample with a value below MDD was defined as 100 pg / ml Ovr110. The minimum detectable dose is defined as two standard abbreviations above the background signal. Most serum samples from healthy patients show low Ovr110 concentrations by sandwich ELISA, while sera from ovarian cancer patients have increased levels of Ovr110.

Ovr110 concentrations were tested in over 2700 serum samples from patients with lung, breast, colon, prostate or ovarian cancer or non-cancerous benign disease. See Table 10 below for a complete list of all samples tested.

FIG. 17 shows Ovr110 concentration in sera from 540 healthy donors and more than 1200 cancer patients. While increased Ovr110 levels are observed in some patients in all cancer types, ovarian cancer patients have the greatest median Ovr110 concentration.
Ovr110 concentrations in the serum of 147 women with serous or endometrial ovarian cancer and 67 sera of women with mucinous cancer were tested with sera representing all four stages of tumor progression. As shown in FIG. 18, the first two ovarian cancer types were positive for Ovr110 by IHC, while not for mucinous cancer. Consistent with these data, the median Ovr110 concentration in the formation of patients with endometrial and serous ovarian cancer is higher than in mucinous cancer patients.

Compared to healthy women, the median Ovr110 concentration in serous and endometrial ovarian cancer is more than twice as high. Most women in this group of 260 healthy women are over 50 years old, reflecting the age distribution of women with ovarian cancer. There can be no difference in Ovr110 detection in healthy women before and after menopause. More importantly, an increase in Ovr110 in the sera of 150 women with benign ovarian disease (50 sera for each patient with endometriosis, enlarged ovary and polycystic ovary) is also undetectable. FIG. 19A summarizes the Ovr110 serum ELISA findings as a receiver operating characteristic (ROC) curve for all ovarian cancers, where the area under the curve (AOC) is 0.78. In addition, FIG. 19B is an ROC curve showing the specific utility of the Ovr110 ELISA for detecting serous ovarian cancer, as shown by an AOC as high as 0.8.
Consistent with our observation that Ovr110 is expressed as a cell surface membrane protein, the concentration of Ovr110 in serum is generally very low, even in women with serous cancer. Therefore, the Ovr110 concentration detected in serum from women with serous cancer is lower than 20 ng / ml.

Example 7: Epitope mapping of Ovr110 Epitopes recognized by antibody panels from the A, C and I series were determined by screening for reactivity of overlapping peptides to antibodies through an ELISA-based assay. Twenty-four overlapping peptides were ordered from SynPep (Dublin, CA). Peptides 1-23 were 15 mers and peptide 24 contained 8 amino acids. The peptide sequence started at the N-terminal amino acid G21 of the Ovr110 protein and ended at the C-terminal A258. These peptides extend to the extracellular region of the mature Ovr110 protein from the end of the signal peptide sequence to the beginning of the transmembrane domain. See FIG. Peptides were provided in small aliquots ranging from 1 to 3 mg as stock solutions. Each peptide was diluted 1: 400 with PBS, and 50 μl was added in duplicate to each well on 96-well 4 × Costar plates (# 3690) (Costar Corporation; Cambridge, Mass.) And left overnight. The above-his tagged mammalian Ovr110 full-length protein was used as a positive control on each 96-well plate. The next day, the plates were flicked dry and blocked with TBST 0.5% BSA for about 40 minutes. Anti-Ovr110 antibody (50 μl) was added at 20 μg / ml per well and incubated at room temperature for about 2 hours. The plate was washed 3 times with TBST wash buffer. The secondary conjugate goat anti-mouse IgFc-AP (Pierce, Rockford, IL) was diluted 1: 5000 in TBST / BSA solution and 50 μl was added to each well. The plate was shaken for 2 hours at room temperature. The plate was washed 3 times and 50 μl of substrate was added to each well and incubated for 15 minutes at room temperature. The substrate used was pNPP (1 mg / ml) in 1 × DEA. To visualize the assay, the plate was read on SpectraMaxPlus (Molecular Devices, Sunnyvale, Calif.) At 405 nm using Softmax Pro (Molecular Devices, Sunnyvale, Calif.) And Excel (Microsoft; Seattle, WA) software for analysis. It was.

  Table 11 below summarizes the results of the above anti-Ovr110 antibody peptide binding experiments. Anti-Ovr110 A, C and I series antibodies showed strong specific reactivity with peptides 8, 13, 15 and 16 of Ovr110. Three types of antibodies I2, I3 and I4 showed strong reactivity with peptide 8, and antibody C9.1 was highly reactive with peptide 16. Four antibodies, C7.1, C12.1, C16.1 and I20 did not recognize any peptide, but bind to full length protein expressing Ovr110 and transfected cells as shown above These suggest that they recognize steric epitopes rather than linear epitopes of Ovr110.

The remaining antibodies tested reacted with peptide 13 or 15 or both. For example, A57.1, A72.1, C10.1, C11.1, and I11 antibodies reacted strongly with peptide 13. A7.1, C3.2 and C6.3 antibodies reacted only with peptide 15. The A87.1, C5.3.1, and C17.1 antibodies showed a novel binding pattern in that they bind to both peptides 13 and 15, except that reactivity to peptide 15 is reactive to peptide 13. It was higher.
Using publicly available software, post-translational modifications were predicted for the complete Ovr110 protein and each feature was mapped to a respective peptide.

  As shown in FIG. 20, the italicized region of Ovr110 represents the site where most A, C and I series antibodies bind to an epitope on Ovr110. This region overlaps the IgV (underlined) and IgC (double underlined) regions of the Ovr110 protein. Interestingly, most A, C and I antibodies bind to peptides 13 and 15 which are putative sites for CK2 phosphorylation, tyrosine sulfation, N-glycosylation and PKC phosphorylation (see Table 11 above). . This sequence is also present in the part where the IgV region separates from the IgC region.

As indicated above, these peptide regions appear to be highly immunogenic and are therefore considered to be part of the Ovr110 protein with a high degree of exposure on the surface. Table 12 provides further evidence for this expectation, which shows that most antibodies (A7.1, A87.1, C3.2, C5.3.1 and C6.3) that bind strongly to peptide 15 by ELISA assay. ) Also shows strong binding on the cell surface of SKBr3, a breast cancer cell line. Anti-Ovr110 monoclonal antibodies A57.1 and I20 are unique in that they bind to Ovr110 on these activated T cells, but do not show significant binding to Ovr110 on SKBr3 cells.

  Previous publications showed conserved tertiary structure in B7 family proteins, but few conserved residues. Sica, above. These conserved residues are thought to be more important to the structure of family members than to activity or receptor recognition for binding. Human B7 family member protein B7.1 (Refseq accession: NP_005182), B7.2 (Refseq accession: NP_787058) and Ovr110 configuration using default settings using publicly available ClustalW Version 1.83 software I went there. See FIG. Combining the arrangement of B7 family proteins with the publicly available 3D model of B7.1 (PDBID: 1DR9) showed the position of conserved residues. Therefore, it was possible to determine where the similar region of the Ovr110 peptide is located on the B7.1 three-dimensional model. According to our findings, the residues on peptides 13 and 15 are exposed on the surface of Ovr110 and are therefore easily reachable and immunogenic. This analysis supports the finding that many antibodies raised against the Ovr110 protein show specific reactivity against peptide 13 or 15 or both. Furthermore, based on the above arrangement and the three-dimensional model of B7.1, an antibody that binds to the IgV region of Ovr110 (residues N47 to F150, see FIG. 20) inhibits binding of Ovr110 to the Ovr110 receptor. Is expected. In particular, antibodies that bind to steric epitopes comprising residues on peptide 12 (SEQ ID NO: 31) or peptide 12 are expected to inhibit binding of Ovr110 to its receptor.

  FIG. 22 shows the arrangement of Ovr110 and Ovr110 homologs from mice (Refseq accession: NP_848709), rats (Refseq accession: XP_227553) and xenopaths (Genbank accession: AAH44000). Deployment was done using the default settings using publicly available ClustalW Version 1.83 software. Because the mouse Ovr110 homolog is not immunogenic in the mouse, the difference between human Ovr110 and the mouse homolog is responsible for the immune response and hence the antibody. Overlapping peptides analyzed with the cross-species Ovr110-homolog configuration and the conserved three-dimensional structure between Ovr110 and family members B7.1 and N72 (Sica, supra) more epitopes recognized by A, C and I series antibodies. It is possible to determine with high accuracy.

As indicated above, antibodies I2, I3 and I4 bind to peptide 8, and the key difference between the mouse and human sequences in this region is amino acids 91-94 (SEQD in humans and SQQH in mice. Where glutamine (Q) and histidine (H) are replaced by glutamic acid (E) and aspartic acid (D), respectively. It can therefore be concluded that I2, I3 and I4 are probably specific for the epitope produced by the sequence SEQD (SEQ ID NO: 44).
Antibodies A57.1, A72.1, C10.1, C11.1, C17.1 and I11 are strongly reactive with peptide 13 but not reactive with peptide 14 or 15. The amino acid sequence change from human to mouse occurs at position 155 where isoleucine (I) is replaced by valine (V) (SMPEI to SMPEV), thus these antibodies are driven by the sequence SMPEV (SEQ ID NO: 45). It can be concluded that it is probably specific for the epitope produced.

  Antibodies A7.1, C3.2, C6.3 bind only to peptide 15. When the mouse and human amino acid sequences are compared in this region (SESLR and SETLR, respectively), the serine at position 165 is replaced with threonine. Thus, these antibodies recognize threonine in either peptide 15 or the full-length natural protein, and thus this binds to the PKC phosphorylation site predicted by the TLR sequence (SEQ ID NO: 46) in SETLR or It can be concluded that the phosphorylation can be blocked.

  Antibodies A87.1, C5.3.1 and C17.1 have a unique binding pattern among these series of antibodies as described above. These antibodies bind to peptides 15 and 13, but the binding to peptide 15 is stronger than the binding to peptide 13. The lack of reactivity with peptide 14 suggests that these antibodies are specific for certain residues common to both peptides 13 and 15, one of which is threonine. The A87.1, C5.3.1 and C17.1 antibodies differ from A7.1, C3.2 and C6.3 which do not bind to peptide 13 in that they also bind to peptide 13. This suggests that peptides 13 and 15 run in parallel chains within the three-dimensional protein structure of Ovr110, as expected for the homologous region within Ovr110 family member B7.1. Antibodies A87.1, C5.3.1 and C17.1 are large enough that these F (ab) domains are located across both chains and thus react simultaneously on both chains. However, stronger reactivity towards peptide 15 suggests that the threonine residues of peptide 13 are in a different configuration and are more difficult to bind. Thus, antibodies A7.1, C3.2, and C6.3 either recognize threonine in a different configuration or, as suggested above, recognize more than threonine residues. There must be.

  Antibody C9.1 is the only antibody that recognizes peptide 16. In this region, position 180 is alanine (A) in mice and valine (V) in humans. Therefore, it is concluded that the C9.1 antibody will be specific for the epitope generated by the sequence TVVW (SEQ ID NO: 47).

Example 8: Ovr110 T cell proliferation functional experiment Based on the binding of A, C and I series anti-Ovr110 antibodies to peptides 13 and 15, these sites protect the immune system, especially with respect to maintaining T cell activity. It is considered important. FIG. 23 is a simplified illustration of the complexity of T cell activation. Activation of T cells requires the interaction of several T cell surface proteins with specific ligands found on antigen presenting cells (APCs) such as B cells or dendritic cells.
First and foremost, the T cell receptor (TCR) must act on the antigenic peptide in the context of the major histocompatibility complex (MHC) molecule. This TCR peptide / MHC signal is required for T cell activation, but a second signal is also required, which is called the costimulatory signal. This signal is generated by another set of cell surface molecules called CD28 and its ligands B7.1 and B7.2. T cell proliferation is enhanced through the interaction of CD28 with B7.1 or B7.2 because this interaction produces IL-2 mRNA, which increases IL-2 cytokine production. Thus, IL-2 production can be used as a direct measure of T cell activation and proliferation. On the other hand, when CTLA-4, which is a homolog of CD28, binds to B7.1 or B7.2, IL-2 production is greatly reduced and T cell expansion is restricted.

As we have shown, Ovr110 is found on ovarian and breast cancer tissues and cells, and on activated T cells and other immune cells. To determine the effect of anti-Ovr110 antibodies on human T cells, an IL-2 proliferation assay was performed by measuring the level of IL-2 production by ELISA. In these experiments, T cells were isolated from peripheral blood mononuclear cells obtained from blood (male donor, age 35-55, Stanford University Blood Center), human T cell isolation purchased from Miltenyi (Auburn, CA). Purify using kit. OKT3-producing hybridomas are purchased from ATCC (Manassas, VA) and scaled up and purified in-house. Since T cells can be activated directly by stimulating the CD3ε chain, 96 well plates are coated overnight at 4 ° C. with 50 μl of 3-5 ug / ml OKT3 antibody (anti-CD3) in PBS. The wells are washed 3 times with PBS and 100 μl T cells are added to the wells at 1.5-2 × 10 ⁶ cells / ml. Individual anti-Ovr110 antibodies, ie A57.1 or I20, are added to the T cells in a volume of 50 μl to measure the effect of the antibodies and IL-2 production by the T cells using an ELISA kit (Roche, Emeryville, Calif.). To make a decision. See the schematic diagram of FIG. The decrease in IL-2 production occurs only by the addition of anti-Ovr110 antibody when an antibody that specifically binds to Ovr110 on T cells or an antibody that binds to peptide 13 of Ovr110, eg, A57.1 or I20, It is useful for inhibiting cell and T cell immune activity. These results are illustrated as scenario 1 in FIG.

  These anti-Ovr110 antibodies are useful for the treatment to reduce the undesirable immune response that occurs in most autoimmune diseases, examples of which include multiple sclerosis, myasthenia gravis, autoimmunity Neurological disorders such as Guillain Valley, autoimmune uveitis, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, autoimmune hepatitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, side Cranial arteritis, antiphospholipid syndrome, vasculitis such as Wegener's granulomatosis, Behcet's disease, psoriasis, herpes zosteritis, pemphigus vulgaris, vitiligo, type I or immune-mediated diabetes, Graves' disease, Hashimoto's thyroiditis, self Immune ovitis and orchitis, autoimmune diseases of the adrenal gland, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, polymyositis, dermatomyositis, spondyloarthropathy such as ankylosing spine Flame, and Sjogren's syndrome, and the like.

  Anti-Ovr110 antibodies that recognize only tumor cells, such as C3.2, A7.1 or C6.3, are used in the IL-2 proliferation assay as described above. Selective binding of Ovr110 to tumor cells indicates that T cells can remain functional by blocking the binding of Ovr110 receptor to Ovr110 ligand on tumor cells. Experiments were performed as described above, except that in addition, SKBr3 cells were added to the wells. If SKBr3 expresses Ovr110 ligand but does not express a receptor (determined by staining experiments with Ovr110-Igfc fusion protein), the effect of SKBr3 tumor cells on activated T cells can be determined. This interaction results in inhibition of T cells because SKBr3 expresses Ovr110 ligand and T cells express a receptor that recognizes Ovr110. See Sica, supra and Prasad, supra. However, the addition of anti-Ovr110 antibodies that specifically bind Ovr110 on tumor cells to the wells does not affect T cells as measured by continued IL-2 production. These results are illustrated in scenario 2 of FIG. These anti-Ovr110 antibodies, such as C3.2, A7.1 or C6.3, bind Ovr110 on tumor cells but do not inhibit the immune response and do not negatively regulate the immune system, breast cancer, ovary The ability to target cancer or tumor cells of cancer or pancreatic cancer has great therapeutic utility.

Example 9: Performance of Ovr110 antibodies in antibody-dependent cytotoxicity functional experiments In addition, anti-Ovr110 tumor specific antibodies such as C3.2, A7.1 or C6.3 exhibit antibody-dependent cytotoxicity (ADCC) Natural killer (NK) cells can be used as effector cells to mediate. This is shown by labeling SKBr3 cells with ⁵¹ chromium for 1 hour. Excess ⁵¹ Cr is washed away and tumor cells are pre-incubated with anti-Ovr110 tumor specific antibodies such as C3.2 or C6.3 with negative and positive control cells at 2 μg / ml for 15-30 minutes To do. Natural killer cells are titrated into 96-well plates in effectors to target ratios ranging from 100: 1 to 0.4415: 1. Incubated tumor cells are added in a constant amount of 10,000 cells per well. Wells containing only tumor cells provide a level of spontaneous lysis. Maximum lysis possible is determined by adding 1% Triton-X diluted in PBS to a set of tumor cells without any effector cells. All wells are performed in triplicate.

After a 4 hour incubation time at 37 ° C., the plates are spun down at 1000 rpm for 5 minutes. The supernatant (100 μl) is collected and read on a gamma counter. Specific lysis is determined by the following equation:
(Experimental value-Spontaneous value) (Triton X treatment-Spontaneous value) x 100
Anti-Ovr110 antibodies that have higher specific lysis than control antibodies are therapeutically useful, and they abolish tumors in the body by stimulating the immune system and using the body's own effector cells. Results from ADCC in vitro assays showing the utility of anti-Ovr110 antibodies in vitro suggest that these antibodies have utility in promoting ADCC in vivo.

Example 10: Deposit
Cell Lines and DNA Deposits The following hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA and have been assigned accession numbers.
The name of the deposited hybridoma cell line can be shortened for convenience of reference. For example, A57.1 corresponds to Ovr110.A57.1. These hybridomas correspond to the clones listed in Table 13 (along with their full names).

  These deposits were made under the provisions of the Budapest Treaty concerning the international recognition of the deposit of microorganisms in patent proceedings and under the provisions of the Budapest Treaty. This ensures the maintenance of viable cultures for 30 years from the date of deposit. The organism has been made available by the ATCC under the terms of the Budapest Treaty and in accordance with the agreement between diaDexus, Inc. and ATCC, which may be issued by the relevant US patent or any US or foreign patent application The first public release of any of the following, guarantees the permanent and unrestricted public availability of the progeny of the culture, and the rules of the Secretary in 35 USC § 122 and in accordance with it Guarantee the availability of offspring to those determined to be entitled by the US Patent and Trademark Director in accordance with (including 37 CFR §1.14 with specific reference to 886 OG 638) To do.

  Applicants of the present application will be able to notify the viable culture of the same culture if the deposited culture is killed, lost or destroyed when cultured under suitable conditions. Agrees to immediately exchange for a new specimen. The availability of the deposited strain should not be considered as a license for the practice of the present invention, in violation of the rights granted under any governmental power in accordance with the patent law. Making these deposits in no way acknowledges that such deposits are necessary to enable the present invention.

It is a figure which shows the result of the FACS analysis of Ovr110 transfected mouse | mouth LMTK cell. FIG. 6 shows immunofluorescence with Ovr110-A57.1 in living ovarian cancer cells and breast cancer cells. FIG. 5 shows Ovr110-A57.1 binding and internalization in living ovarian and breast cancer cells. It is a figure which shows the immunohistochemistry by Ovr110-A57.1 in ovarian serous adenocarcinoma. It is a figure which shows the immunohistochemistry by Ovr110-A57.1 in a breast invasive ductal carcinoma. It is a figure which shows the immunohistochemistry by Ovr110-A57.1 in pancreatic adenocarcinoma. FIG. 3 shows the expression of B7 family members on day 3 in CD3 FITC-gated PHA-stimulated T cells. It is a figure which shows the coupling | bonding of BTLA-Fc fusion protein to the Ovr110-293F cell. FIGS. AC are Western blot detection of Ovr110 protein by mAb A57.1 in cell lines and human tumor tissues.

FIG. 3 shows that Ovr protein is not detected in extracts of major organs. FIG. 5 shows specific knockdown of Ovr110 mRNA in SKBR3 breast cancer cells. It is a figure which shows the down-regulation of Ovr110 protein by siRNA in SKBR3 cell. FIG. 5 shows that knockdown of Ovr110 mRNA induces apoptosis in SKBR3 cells. FIG. 5 shows that knockdown of Ovr110 mRNA induces caspase activity in SKBR3 cells. FIG. 3 shows that overexpression of Ovr110 enhances tumor xenograft growth. FIG. 4 shows that overexpression of Ovr110 protects against apoptosis. FIG. 5 shows Ovr110 epitope maps for different antibodies.

FIG. 5 shows Ovr110 detection in the sera of healthy donors and cancer patients. FIG. 5 shows Ovr110 detection of different types of ovarian cancer and benign disease samples. FIG. 5 is a diagram illustrating a receiver operating characteristic (ROC) curve for Ovr110 detection. Ovr110 protein domain and antibody binding region are shown, including the signal peptide, IgV, IgC and transmembrane region, and the region used to construct overlapping peptides. It is a figure which shows arrangement | positioning of Ovr110 protein and a human family member. It is a figure which shows arrangement | positioning of Ovr110 protein and a homolog. FIG. 2 shows ligand-receptor interaction between T cells and APC. It is a schematic diagram of an Ovr110T cell proliferation function experiment.

Claims (31)

  Is an isolated Ovr110 monoclonal antibody that binds to Ovr110 on a mammalian cell, produced by a hybridoma selected from the group consisting of American Type Culture Collection accession numbers PTA-6266, PTA-7128, and PTA-7129? Or competes with a monoclonal antibody produced by a hybridoma selected from the group consisting of American Type Culture Collection Accession Nos. PTA-6266, PTA-7128 and PTA-7129 and is internalized upon binding to Ovr110 on mammalian cells , Said antibody.   2. The antibody of claim 1, which is a chimeric, humanized or human antibody, or antibody fragment thereof.   2. The antibody of claim 1, wherein the antibody binds to an Ovr110 peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 and 34.   4. The antibody of claim 3, wherein the Ovr110 peptide comprises a post-translational modification.   2. The antibody of claim 1, wherein the antibody binds to an epitope consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45 and 46.   2. The antibody of claim 1 conjugated to a growth inhibitory agent or cytotoxic agent.   The antibody of claim 6, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   The antibody according to claim 1, wherein the mammalian cell is a cancer cell.   An anti-Ovr110 monoclonal antibody that inhibits the growth of Ovr110-expressing cancer cells in vivo and is produced by a hybridoma selected from the group consisting of American Type Culture Collection accession numbers PTA-6266, PTA-7128 and PTA-7129.   An antibody which is a chimeric or humanized antibody of the antibody of claim 9.   An antibody which is a humanized form of the antibody of claim 9.   10. The antibody of claim 9, wherein the cancer cell is from a cancer selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer and breast cancer.   Hybridoma cells deposited under American Type Culture Collection accession numbers PTA-6266, PTA-7128 and PTA-7129.   A composition comprising the antibody according to claim 1 or 9 and a carrier.   15. The composition of claim 14, wherein the antibody is conjugated to a cytotoxic agent.   A composition for killing Ovr110-expressing cancer cells, comprising the antibody according to claim 1, wherein the cancer cell is contacted with the antibody, thereby killing the cancer cell. Said composition.   The composition according to claim 16, wherein the cancer cells are selected from the group consisting of ovarian cancer cells, pancreatic cancer cells, lung cancer cells and breast cancer cells.   18. The composition according to claim 17, wherein the ovarian cancer or breast cancer is ovarian serous adenocarcinoma or breast invasive ductal carcinoma.   18. A composition according to claim 17, wherein the cancer cells are from metastatic ovarian cancer, pancreatic cancer, lung cancer or breast cancer.   A composition for ameliorating an Ovr110-expressing cancer in a mammal, comprising the antibody of claim 9, wherein a therapeutically effective amount of the antibody is administered to the mammal. object.   21. The composition of claim 20, wherein the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, lung cancer and breast cancer.   22. The composition of claim 21, wherein the ovarian cancer or breast cancer is ovarian serous adenocarcinoma or breast invasive ductal carcinoma.   21. The composition of claim 20, wherein the antibody is administered in combination with at least one chemotherapeutic agent.   24. The composition of claim 23, wherein the chemotherapeutic agent is paclitaxel or a derivative thereof.   An article of manufacture comprising a container and a composition contained therein, wherein the composition comprises the antibody of claim 1.   26. The article of manufacture of claim 25, further comprising a package insert indicating that it can be used to detect or treat ovarian cancer, pancreatic cancer, lung cancer or breast cancer. A method of detecting Ovr110 overexpression in a subject in need thereof, comprising:
(A) mixing the sample of interest with the Ovr110 antibody of claim 1 under conditions suitable for specific binding of the Ovr110 antibody to Ovr110 in the sample;
(B) determining the level of Ovr110 in the sample;
(C) comparing the level of Ovr110 determined in step b with the level of Ovr110 in the control, wherein an increase in the level of Ovr110 in the sample from the subject compared to the control is Wherein said method shows Ovr110 overexpression in   The target sample is body fluid, cell, cancer cell, blood, plasma, serum, urine, saliva, sputum, tears, ascites, peritoneal washing, lymph, bile, semen, pus, amniotic fluid, aqueous humor, earwax, wrinkles, sputum 28. The method of claim 27, selected from the group consisting of: intestinal fluid, menstruation, milk, mucus, pleural fluid, sweat, vaginal lubricating fluid, vomiting, cerebrospinal fluid, and synovial fluid.   28. The method of claim 27, wherein the subject is ovarian serous adenocarcinoma, breast invasive ductal carcinoma or a metastatic cancer thereof.   30. The method of claim 28, wherein the control is a serum sample from a subject who does not have a cancer that overexpresses Ovr110. A method for screening an antibody that binds to an epitope bound by the antibody of claim 1, comprising:
(A) combining a sample containing Ovr110 with a test antibody and the antibody of claim 1 to form a mixture;
(B) determining the level of Ovr110 antibody bound to Ovr110 in the mixture, and (c) comparing the level of Ovr110 antibody bound in the mixture of step (a) to the control mixture;
Wherein the level of Ovr110 antibody that binds to Ovr110 in the mixture compared to a control is an indication of the binding of the test antibody to the epitope bound by the anti-Ovr110 antibody of claim 1. Said screening method.

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